In vivo gene silencing by chemically modified and stable siRNA

ABSTRACT

The present invention provides compositions for RNA interference and methods of use thereof. In particular, the invention provides small interfering RNAs (siRNAs) having modification that enhance the stability of the siRNA without a concomitant loss in the ability of the siRNA to participate in RNA interference (RNAi). The invention also provides siRNAs having modification that increase targeting efficiency. Modifications include chemical crosslinking between the two complementary strands of an siRNA and chemical modification of a 3′ terminus of a strand of an siRNA. Preferred modifications are internal modifications, for example, sugar modification, nucleobase modification and/or backbone modifications. Such modifications are also useful, e.g., to improve uptake of the siRNA by a cell. Functional and genomic and proteomic methods are featured. Therapeutic methods are also featured.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/661,810, filed Mar. 18, 2015, which is a continuation of U.S. patentapplication Ser. No. 13/561,357, filed Jul. 30, 2012, now U.S. Pat. No.9,012,623, which is a continuation of U.S. patent application Ser. No.10/672,069, filed Sep. 25, 2003, which claims the benefit of: U.S.Provisional Patent Application Ser. No. 60/413,529, filed Sep. 25, 2002;U.S. Provisional Patent Application Ser. No. 60/426,982, filed Nov. 15,2002; U.S. Provisional Patent Application Ser. No. 60/458,051, filedMar. 26, 2003; and U.S. Provisional Patent Application Ser. No.60/493,095, filed Aug. 5, 2003. The entire contents of theabove-referenced patent applications are incorporated herein by thisreference.

BACKGROUND OF THE INVENTION

RNA interference (RNAi) is the process whereby double-stranded RNA(dsRNA) induces the sequence-specific degradation of homologous mRNA.Although RNAi was first discovered in Caenorhabditis elegans (Fire etal., 1998), similar phenomena had been reported in plants(post-transcriptional gene silencing [PTGS]) and in Neurospora crassa(quelling) (reviewed in Hammond et al., 2001; Sharp, 2001). It hasbecome clear that dsRNA-induced silencing phenomena are present inevolutionarily diverse organisms, e.g., nematodes, plants, fungi andtrypanosomes (Bass, 2000; Cogoni and Macino, 2000; Fire et al., 1998;Hammond et al., 2001; Ketting and Plasterk, 2000; Matzke et al., 2001;Sharp, 2001; Sijen and Kooter, 2000; Tuschl, 2001; Waterhouse et al.,2001). Biochemical studies in Drosophila embryo lysates and S2 cellextracts have begun to unravel the mechanisms by which RNAi works(Bernstein et al., 2001; Tuschl et al., 1999; Zamore et al., 2000).

RNAi is initiated by an ATP-dependent, processive cleavage of dsRNA into21- to 23-nucleotide (nt) short interfering RNAs (siRNAs) (Bernstein etal., 2001; Hamilton and Baulcombe, 1999; Hammond et al., 2000; Zamore etal., 2000) by the enzyme Dicer, a member of the RNase III family ofdsRNA-specific endonucleases (Bernstein et al., 2001). These nativesiRNA duplexes containing 5′ phosphate and 3′ hydroxyl termini are thenincorporated into a protein complex called RNA-induced silencing complex(RISC) (Hammond et al., 2000). ATP-dependent unwinding of the siRNAduplex generates an active complex, RISC* (the asterisk indicates theactive conformation of the complex) (Nykanen et al., 2001). Guided bythe antisense strand of siRNA, RISC* recognizes and cleaves thecorresponding mRNA (Elbashir et al., 2001b; Hammond et al., 2000;Nykanen et al., 2001).

Recently, Tuschl and colleagues (Elbashir et al., 2001a) havedemonstrated that RNAi can be induced in numerous mammalian cell linesby introducing synthetic 21-nt siRNAs. By virtue of their small size,these siRNAs avoid provoking an interferon response that activates theprotein kinase PKR (Stark et al., 1998). Functional anatomy studies ofsynthetic siRNA in Drosophila cell lysates have demonstrated that eachsiRNA duplex cleaves its target RNA at a single site (Elbashir et al.,2001c). The 5′ end of the guide siRNA sets the ruler for defining theposition of target RNA cleavage (Elbashir et al., 2001c). 5′phosphorylation of the antisense strand is required for effective RNAinterference in vitro (Nykanen et al., 2001). Mutation studies haveshown that a single mutation within the center of an siRNA duplexdiscriminates between mismatched targets (Elbashir et al., 2001c). Theseexperiments showed a more stringent requirement for the antisense strandof the trigger dsRNA as compared to the sense strand (Grishok et al.,2000; Parrish et al., 2000). Notably these phenomena were demonstratedin vitro or in cell culture systems.

There is a need for further study of such systems. Moreover, thereexists a need for the development of reagents suitable for use in vivo,in particular for use in developing human therapeutics.

SUMMARY OF THE INVENTION

The present invention is based on the suprising discovery that siRNAmolecules (i.e., duplex siRNA molecules) can be modified at internalresidues such that properties important for in vivo applications, inparticular, human therapeutic applications, are improved withoutcompromising the RNAi activity of the siRNA molecules. In particular,the invention is based on the discovery of modifications which aretolerated in siRNA molecules, modifications which are not tolerated, andthree-dimensional structural features that are or are not required inorder for siRNA molecules to mediate RNAi. Accordingly, the presentinvention provides compositions for RNA interference and methods of usethereof. In particular, the invention provides small interfering RNAs(siRNAs) having modification or combination of modifications thatenhance the stability of the siRNA without a comcommittent loss in theability of the siRNA to participate in RNA interference (RNAi). Theinvention also provides siRNAs having modification that increasetargeting efficiency.

Modifications include chemical crosslinking between the twocomplementary strands of an siRNA and chemical modification of a 3′terminus of a strand of an siRNA. Preferred modifications are internalmodifications, for example, sugar modifications, nucleobasemodifications and/or backbone modifications. Such modifications are alsouseful to improve uptake of the siRNA by a cell. Functional and genomicand proteomic methods are featured. Therapeutic methods are alsofeatured.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-D depicts a dual fluorescence reporter assay system for RNAianalysis in HeLa cells. (A) Graphical representation of dsRNAs used fortargeting GFP mRNA and RFP mRNA. GFP and RFP were encoded by thepEGFP-C1 and pDsRed1-N1 reporter plasmid, respectively. siRNAs weresynthesized with 2-nt deoxythymidine overhangs at the 3′ end. Theposition of the first nucleotide of the mRNA target site is indicatedrelative to the start codon of GFP mRNA or RFP mRNA. The sequence of theantisense strand of siRNA is exactly complementary to the mRNA targetsite. (B) Fluorescence images showing specific RNA interference effectsin living HeLa cells. Fluorescence in living cells was visualized byfluorescence microscopy at 48 hours post transfection. Panels a and b,images of mock-treated cells (no siRNA added); panels c and d, images ofGFP siRNA-treated cells; panels e and f, images of RFP siRNA-treatedcells. (C) Quantitative analysis of RNAi effects in HeLa cells.Fluorescence emission spectra of GFP and RFP in total cell lysates weredetected by exciting at 488 nm and 568 nm, respectively. (D) Kinetics ofRNAi effects in HeLa cells. Ratios of normalized GFP to RFP fluorescenceintensity over a 66-hour time course. The fluorescence intensity ratioof target (GFP) to control (RFP) protein was determined in the presenceof double strand (ds) RNA (green bars) and normalized to the ratioobserved in the presence of antisense strand (as) RNA (blue bars).Normalized ratios less than 1.0 indicate specific RNA interference.Maximal RNAi effect occurred at 42 hours post transfection.

FIG. 1E-F depicts analysis of specific RNAi activities by Westernblotting. Antisense and double strand RNA are indicated as as and ds,respectively. GFP as (E, left panel), GFP ds (E, right panel), RFP as(F, left panel) or RFP ds (F, right panel) were cotransfected withpEGFP-C1 and pDsRed1-N1 reporter plasmids into HeLa cells. Cells wereharvested at various times, resolved on 10% SDS-PAGE, transferred ontoPVDF membranes, and immunoblotted with antibodies against EGFP andDsRed1-N1. The membrane was stripped and re-probed with anti-actinantibody to check for equal loading of total proteins.

FIG. 1G depicts expression of GFP in HeLa cells treated with antisenseor double-stranded siRNA targeting GFP. Transfected cells were harvestedat various times after transfection and total cell lysates were analyzedby fluorescence spectroscopy. Fluorescence emission spectra of GFP andRFP were detected by exciting at 488 nm and 568 nm, respectively.

FIG. 2A-B depicts the modification of GFP siRNA duplexes. (A) Structureof 5′-N3 (amino group with 3-carbon linker, red) and 3′-Pmn (puromycin,blue) modifications. (B) Classification and nomenclature of the modifiedsiRNAs. Sense (top row, purple) and antisense (bottom row, black)strands of siRNA species are shown with their 5′-N3 (red) and 3′-Pmn orbiotin (blue) modifications. A dinucleotide internal bulge structure(green) was introduced in sense, antisense, or duplex RNAs.

FIG. 3A-L depicts fluorescence images showing RNA interference effectsin living HeLa cells transfected with modified siRNA duplexes. HeLacells were cotransfected by lipofectamine with pEGFP-C1, pDsRed1-N1reporter plasmids and siRNA with a 5′ modification (panels C, D, and E),3′ modification (panels F, G, H, and I) or internal bulge (panels J, K,and L). Fluorescence in living cells was visualized at 48 hours posttransfection. GFP fluorescence (left panels) and phase contrast images(right panels) are shown. RNA used in each experiment is indicated onthe left of each pair of panels.

FIG. 4A-B depicts quantitative analysis of RNAi effects in HeLa cellstransfected with modified siRNAs. pEGFP-C1 (as reporter), pDsRed1-N1 (ascontrol) plasmids and 50 nM siRNA were cotransfected into HeLa cells bylipofectamine. Cells were harvested at various times after transfection.Fluorescence emission spectra of GFP and RFP in total cell lysates weredetected by exciting at 488 nm and 568 nm, respectively. (A) GFPemission spectra of modified siRNAi-treated cells. Emission spectra ofGFP in lysates from cells transfected with 5′-modified GFP siRNAs (upperpanel), 3′-modified GFP siRNAs (middle panel) and bulge-containing GFPsiRNAs (lower panel). For comparison, results from antisense- (as, redline) and unmodified duplex siRNA (ds, black line)-treated cells areincluded in each panel. (B) Ratios of normalized GFP to RFP fluorescenceintensity in lysates from modified siRNA-treated HeLa cells over 66hours. The fluorescence intensity ratio of target (GFP) to control (RFP)fluorophore was determined in the presence of 5′-modified GFP siRNAs(upper panel), 3′-modified GFP siRNAs (middle panel), andbulge-containing GFP siRNAs (lower panel) and normalized to the ratioobserved in the presence of antisense strand siRNA. Normalized ratiosless than 1.0 indicate specific RNA interference effects. Forcomparison, results from antisense RNA and duplex siRNA-treated cellsare included in each panel (as, orange bars; ds, yellow bars).

FIG. 5 depicts the isolation of 5′ end phosphorylated and 3′ endbiotinylated siRNA from HeLa cells. HeLa cells were cotransfected withbiotinylated GFP duplex siRNA (ss/as3′-Biotin) and pEGFP-C1 plasmid asdescribed in Experimental Procedures. The siRNA was isolated by pull outassay and subjected to phosphatase and kinase reactions (seeExperimental Procedures). Briefly, streptavidin magnetic beads were usedto pull out biotinylated siRNAs from transfected cells, washed to removeunbound RNA, and split into two aliquots. One aliquot wasdephosphorylated with shrimp alkaline phosphatase (SAP), and the RNA 5′ends labeled with ³²P by T4 polynucleotide kinase (PNK) reaction. Theother aliquot was not dephosphorylated. RNA was resolved on 20%polyacrylamide-7M Urea gels and visualized by phosphorimager analysis.Lanes 1-3 (marker lanes) contain 5′-end-labeled RNA: lane 1, sensestrand (ss); lane 2, 3′ biotinylated antisense strand (as3′-Biotin);lane 3, heat denatured (10 min at 95° C.) siRNA duplex (ss/as3′-Biotin).Lanes 5-14, isolated biotinylated siRNA with SAP treatment (lanes 5-9)or without (lanes 10-14). Lane 4, RNA isolated as above from HeLa cellswithout siRNA transfection.

FIG. 6A-E depicts RNA interference activities of covalentlyphotocross-linked duplex RNA in HeLa cells. (A) Structure of a psoralenderivative, 4′-hydroxymethyl-4,5′,8-trimethylpsoralen (HMT), used tocross-link the duplex RNA. (B) Photocross-linking sites in GFP siRNA.Three preferred sites for psoralen addition to a duplex RNA are shown bycyan letters with red bars indicating the C-U cross-links formed by UVirradiation in the presence of HMT. (C) Psoralen photocross-linking ofsiRNA duplexes. Mixtures of siRNA duplex and psoralen were exposed to UV360 nm and denatured. Cross-linked and noncross-linked siRNAs wereresolved on 20% PAGE containing 7 M urea (lanes 2 and 3). UV-irradiatedRNA bands were excised from the gel and purified. Purified cross-linkeddsRNA (ds-XL) and noncross-linked dsRNA (ds*) are shown in lanes 6 and5, respectively. To confirm the nature and purity of the cross-link, aportion of the 360 nm UV-irradiated sample (lane 3) was UV-irradiated at254 nm. Photoreversal of psoralen cross-linked siRNA resulted inproducts with similar electrophoretic mobility to the siRNA duplexwithout HMT treatment (lane 4). (D) Fluorescence images showing RNAinterference effects of psoralen photocross-linked siRNAs in living HeLacells. Purified cross-linked ds siRNA (ds-XL, bottom panels) wascotransfected with reporter pEGFP-C1 and control pDsRed1-N1 plasmidsinto HeLa cells for dual fluorescence reporter assays. Fluorescence(left panels) and phase contrast (right panels) images of living cellswere taken 48 hours post transfection. For comparison, images fromnoncross-linked ds siRNA (ds*, middle panels) and antisense siRNA (as,top panels) are also shown. (E) GFP emission spectra of psoralenphotocross-linked siRNA duplex-treated cells. Cell lysates were preparedfrom HeLa cells treated with antisense siRNA (as), unmodifiedUV-irradiated duplex siRNA (ds*) and cross-linked ds siRNA (ds-XL) andanalyzed by fluorescence spectroscopy. Fluorescence emission spectra ofGFP and RFP were detected by exciting at 488 nm and 568 nm,respectively. GFP emission spectra are shown normalized to RFPexpression.

FIG. 7 depicts the isolation of psoralen-cross-linked siRNA from humancells. siRNA duplexes were conjugated with 3′ biotin (ss/as3′-Biotin),psoralen cross-linked and purified as described in FIG. 6 and inExperimental Procedures. HeLa cells were cotransfected by lipofectaminewith cross-linked siRNA (ss/as3′-Biotin-XL) and pEGFP-C1 plasmid, andsiRNA were isolated by biotin pull out assay at 30 h post transfectionas described in Experimental Procedures. Briefly, streptavidin-magneticbeads with biotinylated siRNA were subjected to phosphatase treatmentand 5′ end-labeled with ³²P. RNA was resolved on 20% polyacrylamide-7Murea gels and visualized by phosphorimager analysis. Lane 1, RNA fromHeLa cells without siRNA transfection. Lane 2, ³²P-labelednoncross-linked siRNA duplex (ss/as3′-Biotin). Lane 3, ³²P-labeled 3′biotinylated anti-sense strand siRNA (as3′-Biotin). Lane 4, ³²P-labeledsense strand RNA (ss). Lane 5, ³²P-labeled cross-linked siRNA duplex(ss/as3′-Biotin-XL). Lanes 7 and 8, siRNA isolated from HeLa cellstreated with cross-linked siRNA duplex (ss/as3′-Biotin-XL). Lanes 6 and8, UV-irradiation (254 nm) of cross-linked siRNA to photoreverse thepsoralen cross-links.

FIG. 8A-B depicts fluorescence intensity spectra for extracts of cellstransfected with various GFP- and/or RFP-encoding plasmids and,optionally, treated with siRNAs targeting GFP and/or RFP mRNAs. (A)depicts the fluorescence intensity spectra for extracts from cellstransfected with dsRed1-N1 versus dsRed2-N1. (B) depicts RNAi of GFP orRFP, left and right panels, respectively.

FIG. 9A-B depicts a quantitative analysis of RNAi effects in HeLa cellstransfected with modified single-stranded (antisense strand) siRNAs. (A)depicts the fluorescence intensity ratio of target (GFP) to control(RFP) fluorophore. (B) depicts the fluorescence intensity ratio oftarget (GFP) to control (RFP) fluorophore in the presence of variousamount of 5′-phosphorylated as siRNA.

FIG. 10A-E depicts a quantitative analysis of RNAi effects in HeLa cellstransfected with modified duplex siRNAs. (A) depicts the results fromcells treated with duplex siRNA with 2′-Deoxy modification at internalresidues within the sense strand (ss-2′Deoxy/as, lanes 6-11). (B)depicts results from cells treated with duplex siRNA with 2′-O-Methylmodification at internal residues within the sense strand (ss-2′Ome/as,lanes 6-11) or the antisense strand (ss/as-2′-Ome, lanes 12-17). (C)depicts dual fluorescence assay data of 2′FU, FC siRNAs, modified onlyin the sense strand (ss-2′FU, 2′-FC/as, lanes 6-15), only in theantisense strand (ss/as-2′-FU,2′-FC, lanes 16-25), or in both strands(ds-2′FU,2′FC, lanes 26-35). (D) depicts results from cells treated withduplex siRNA with phosphorothiolate modification at each backboneresidue of the sense strand (ss-P-S-all/as, lanes 6-12), antisensestrand (ss/as-P-S-all, lanes 13-22) and both strands (ds-P-S-all, lanes23-31). (E) depicts results from cells treated with duplex siRNA withphosphorothiolate modification at each backbone residue of both strandsexcept for bases 9-12 of the antisense strand (ds-P-S, except centerregion, lanes 15-23).

FIG. 11 depicts the kinetics of RNAi effects of duplex siRNA with2′-Fluoro uridine and cytidine modification in HeLa cells.

FIG. 12A-E depicts the stability of duplex siRNA with 2′-Fluoro uridineand cytidine modification in HeLa cell lysates. (A) depicts a stabilitycomparison of unmodified and modified antisense strand siRNA. (B)depicts a stability comparison of duplex siRNAs with unmodified andmodified antisense strand. (C) depicts a stability comparison of duplexsiRNAs containing modification at both strands. (D) shows the stabilityof the various 2′FU, FC modified siRNAs as compared to wild type siRNAsover time. (E) depicts the stability of P-S modified EGFP siRNAs.

FIG. 13A-D depicts a quantitative analysis of RNAi effects of duplexsiRNAs with 2′-Fluoro uridine and cytidine modifications, and 2′-Fluorouridine and cytidine modifications in combination with 2′-deoxymodifications, in HeLa cells. (A) depicts modified siRNA duplexes withmodifications in the antisense strand at the 2′ position of the sugarunit. (B) depicts data from cells treated with duplex siRNA withmodified antisense strands. (C) depicts siRNA duplexes withmodifications in both strands at the 2′ position of the sugar unit. (D)results from cells treated with duplex siRNA with modifications in bothstrands as set forth in FIG. 13C.

FIG. 14A-C depicts a quantitative analysis of RNAi effects of duplexsiRNAs with N3-Methyl uridine modifications in HeLa cells. (A) depictsthe structure of N³-Methyl-Uridine (3 mU). (B) RNAi was also abolishedif only one 3 MU modification was introduced specifically at U11 of theantisense strand, which is one of the nucleotides that base pairs withA248 of the target EGFP mRNA cleavage site. (C) depicts the results fromcells treated with duplex siRNA having 3 mU modifications within theentire antisense strand (SS/AS-3 mU, lanes 7-15), 3 mU modificationswithin the entire antisense strand and 2′-Fluoro modifications aturidine and cytidine bases within the sense strand (SS-2′FU, FC/AS-3 mU,lanes 16-24), and 3 mU modification at position 11 within the antisensestrand (SS/AS-(11)-3 mU, lanes 25-33).

FIG. 15A-C depicts a quantitative analysis of RNAi effects of duplexsiRNAs with 2-nucleotide mismatches in the antisense strand in HeLacells. (A) depicts results suggesting that a single cross-linking eventoccurring near the 3′ end of the antisense strand still allowed for theinitial unwinding of duplex siRNAs from the 5′ end, freeing enough ofthe nucleotides in the antisense strand to hybridize to the target mRNAand induce RNAi, even if unwinding was not complete. The location ofthis crosslinking site is indicated by a bar in FIG. 15A. (B) depictsEGFP siRNAs with mismatched base pairs at either the 5′ (nt 1, 2) or 3′(nt 18, 19) ends were introduced into the antisense strand. (C) depictsdata of Example XIII, wherein 2′FU, FC plus dATPs, dGTPs wereincorporated into the antisense strand siRNAs predominantly at the 5′end (nts 1-13) or predominantly at the 3′ end (nts 9-19).

FIG. 16 depicts a quantitative analysis of RNAi effects of duplex siRNAswith 5-Br uridine, 5-I uridine and diaminopurine modifications in theantisense strand in HeLa cells.

FIG. 17 depicts target RNA cleavage by duplex siRNAs with variousmodifications in HeLa cell lysates.

FIG. 18 depicts the mechanism for RNAi in human cells highlighting therequirement of the A-form helix and major groove for mRNA cleavage andthe steps which do not require the RNA 2′OH of the guide antisensesiRNA.

FIG. 19A-B depicts the structures of EGFP siRNA and the structure andnomenclature of preferred chemical modifications. (A) depicts thestructures of EGFP siRNA. (B) depicts the structure and nomenclature ofpreferred chemical modifications.

FIG. 20 is a drawing of the structure of a novel photocleavable biotin.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the suprising discovery that siRNAmolecules (i.e., duplex siRNA molecules) can be modified at internalresidues such that properties important for in vivo applications, inparticular, human therapeutic applications, are improved withoutcompromising the RNAi activity of the siRNA molecules. The instantinvention features siRNAs having significant modification to internalresidues within the siRNA, providing new rules for designing effectiveand stable siRNAs for RNAi-mediated gene-silencing applications. Mostremarkably, modifications at the 2′ position of pentose sugars in siRNAsshowed that 2′OH groups are not required for RNAi, indicating that theRNAi machinery does not require the 2′OH for recognition of siRNAs andthat catalytic ribonuclease activity of RNA-induced silencing complexes(RISC) does not involve the 2′OH of the guide antisense RNA. In fact,the instant inventor was able to replace an entire siRNA strand with 2′deoxy- and 2′ fluoro-nucleotides and still induce RNAi in human cells.

This is a significant finding for several reasons. First, it indicatesthat, mechanistically, the RNAi machinery does not require the 2′ OH forrecognition of siRNAs and that the catalytic ribonuclease activity ofRISC does not involve 2′ OH groups of the guide antisense RNA. This alsomeans that a variety of chemical groups, including fluoro- ordeoxy-groups, could substitute for the 2′OH in siRNAs and that nodistinguishing chemical specificity was required for RNAi at the 2′position. This finding now directs attention to core structuralelements, like the A-form helix and the major groove formed by theA-form helix at the cleavage site and not RNA itself, as being theessential determinants of RNAi. These findings are particularly usefulin the design of effective siRNAs. It also explains why DNA-DNA orDNA-RNA hybrids are not recognized for RNAi. Differences between themiRNA-induced silencing mechanism and siRNA-mediated RNAi are furtherexplained by these results in that what distinguishes whether one isinduced over the other is the structure of the RNA-RNA helix. Stillanother important implication of these results is that alternatechemical groups at the 2′ position that allow the A-form helix to beretained but help siRNAs evade recognition by RNases increased siRNAstability and prolonged RNAi effects induced in vivo.

Such modifications have the added feature of enhancing properties suchas cellular uptake of the siRNAs and/or stability of the siRNAs.Preferred modifications are made at the 2′ carbon of the sugar moiety ofnucleotides within the siRNA. Also preferred are certain backbonemodifications, as described herein. Also preferred are chemicalmodifications that stabilize interactions between base pairs, asdescribed herein. Combinations of substitution are also featured.Preferred modifications maintain the structural integrity of theantisense siRNA-target mRNA duplex. Methods of mediating RNAi inmammals, preferably humans, are featured as are kits for suchtherapeutic use.

The present invention features modified siRNAs. siRNA modifications aredesigned such that properties important for in vivo applications, inparticular, human therapeutic applications, are improved withoutcompromising the RNAi activity of the siRNA molecules e.g.,modifications to increase resistance of the siRNA molecules tonucleases. Modified siRNA molecules of the invention comprise a sensestrand and an antisense strand, wherein the sense strand or antisensestrand is modified by the substitution of at least one nucleotide with amodified nucleotide, such that, for example, in vivo stability isenhanced as compared to a corresponding unmodified siRNA, or such thatthe target efficiency is enhanced compared to a corresponding unmodifiedsiRNA. Such modifications are also useful to improve uptake of the siRNAby a cell. Preferred modified nucleotides do not effect the ability ofthe antisense strand to adopt A-form helix conformation whenbase-pairing with the target mRNA sequence, e.g., an A-form helixconformation comprising a normal major groove when base-pairing with thetarget mRNA sequence.

Modified siRNA molecules of the invention (i.e., duplex siRNA molecules)can be modified at the 5′ end, 3′ end, 5′ and 3′ end, and/or at internalresidues, or any combination thereof. Internal siRNA modifications canbe, for example, sugar modifications, nucleobase modifications, backbonemodifications, and can contain mismatches, bulges, or crosslinks. Alsopreferred are 3′ end, 5′ end, or 3′ and 5′ and/or internalmodifications, wherein the modifications are, for example, crosslinkers, heterofunctional cross linkers, dendrimer, nano-particle,peptides, organic compounds (e.g., fluorescent dyes), and/orphotocleavable compounds.

In one embodiment, the siRNA molecule of the invention comprises one ormore (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) endmodifications. Modification at the 5′ end is preferred in the sensestrand, and comprises, for example, a 5′-propylamine group.Modifications to the 3′ OH terminus are in the sense strand, antisensestrand, or in the sense and antisense strands. A 3′ end modificationcomprises, for example, 3′-puromycin, 3′-biotin and the like.

In another embodiment, the siRNA molecule of the invention comprises oneor more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) crosslinks,e.g., a crosslink wherein the sense strand is crosslinked to theantisense strand of the siRNA duplex. Crosslinkers useful in theinvention are those commonly known in the art, e.g., psoralen, mitomycinC, cisplatin, chloroethylnitrosoureas and the like. A preferredcrosslink of the invention is a psoralen crosslink. Preferably, thecrosslink is present downstream of the cleavage site referencing theantisense strand, and more preferably, the crosslink is present at the5′ end of the sense strand.

In another embodiment, the siRNA molecule of the invention comprises oneor more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more)sugar-modified nucleotides. Sugar-modifed nucleotides useful in theinvention include, but are not limited to: 2′-fluoro modifiedribonucleotide, 2′-OMe modified ribonucleotide, 2′-deoxy ribonucleotide,2′-amino modified ribonucleotide and 2′-thio modified ribonucleotide.The sugar-modified nucleotide can be, for example, 2′-fluoro-cytidine,2′-fluoro-uridine, 2′-fluoro-adenosine, 2′-fluoro-guanosine,2′-amino-cytidine, 2′-amino-uridine, 2′-amino-adenosine,2′-amino-guanosine or 2′-amino-butyryl-pyrene-uridine. A preferredsugar-modified nucleotide is a 2′-deoxy ribonucleotide. Preferably, the2′-deoxy ribonucleotide is present within the sense strand and, forexample, can be upstream of the cleavage site referencing the antisensestrand or downstream of the cleavage site referencing the antisensestrand. A preferred sugar-modified nucleotide is a 2′-fluoro modifiedribonucleotide. Preferably, the 2′-fluoro ribonucleotides are in thesense and antisense strands. More preferably, the 2′-fluororibonucleotides are every uridine and cytidine.

In another embodiment, the siRNA molecule of the invention comprises oneor more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more)nucleobase-modified nucleotides. Nucleobase-modified nucleotides usefulin the invention include, but are not limited to: 5-bromo-uridine,5-iodo-uridine, 5-methyl-cytidine, ribo-thymidine, 2-aminopurine,5-fluoro-cytidine, and 5-fluoro-uridine, 2,6-diaminopurine,4-thio-uridine; and 5-amino-allyl-uridine and the like.

In another embodiment, the siRNA molecule of the invention comprises oneor more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more)backbone-modified nucleotides, for example, a backbone-modifiednucleotide containing a phosphorothioate group. The backbone-modifiednucleotide is within the sense strand, antisense strand, or preferablywithin the sense and antisense strands.

In another embodiment, the siRNA molecule of the invention comprises asequence wherein the antisense strand and target mRNA sequences compriseone or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more)mismatches. Preferably, the mismatch is downstream of the cleavage sitereferencing the antisense strand. More preferably, the mismatch ispresent within 1-6 nucleotides from the 3′ end of the antisense strand.In another embodiment, the siRNA molecule of the invention comprises abulge, e.g., one or more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, ormore) unpaired bases in the duplex siRNA. Preferably, the bulge is inthe sense strand.

In another embodiment, the siRNA molecule of the invention comprises anycombination of two or more (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10, ormore) siRNA modifications as described herein. For example, a siRNAmolecule can comprise a combination of two sugar-modified nucleotides,wherein the sugar-modified nucleotides are 2′-fluoro modifiedribonucleotides, e.g., 2′-fluoro uridine or 2′-fluoro cytidine, and2′-deoxy ribonucleotides, e.g., 2′-deoxy adenosine or 2′-deoxyguanosine. Preferably, the 2′-deoxy ribonucleotides are in the antisensestrand, and, for example, can be upstream of the cleavage sitereferencing the antisense strand or downstream of the cleavage sitereferencing the antisense strand. Preferably, the 2′-fluororibonucleotides are in the sense and antisense strands. More preferably,the 2′-fluoro ribonucleotides are every uridine and cytidine.

The invention is also related to the discovery that certaincharacteristics of siRNA are necessary for activity and thatmodifications can be made to an siRNA to alter physicochemicalcharacteristics such as stability in a cell and the ability of an siRNAto be taken up by a cell. Accordingly, the invention includes siRNAderivatives; siRNAs that have been chemically modified and retainactivity in RNA interference (RNAi). The invention also includes a dualfluorescence reporter assay (DFRA) that is useful for testing theactivity of siRNAs and siRNA derivatives.

Accordingly, the invention includes an siRNA derivative that includes ansiRNA having two complementary strands of nucleic acid, such that thetwo strands are crosslinked, a 3′ OH terminus of one of the strands ismodified, or the two strands are crosslinked and modified at the 3′OHterminus. The siRNA derivative can contain a single crosslink (e.g., apsoralen crosslink). In some embodiments, the siRNA derivative has abiotin at a 3′ terminus (e.g., a photocleavable biotin such as the novelphotocleavable biotin of FIG. 8), a peptide (e.g., a Tat peptide), ananoparticle, a peptidomimetic, organic compounds (e.g., a dye such as afluorescent dye), or dendrimer.

The invention also includes a method of inhibiting expression of an RNA.The method includes the steps of introducing into a cell an siRNAderivative such as those described herein, and such that the siRNAderivative is targeted to the RNA.

The invention also includes a method that includes the step ofcontacting a cell with a concentration of an siRNA derivative sufficientto inhibit expression of a target gene. In some embodiments, the siRNAderivative is a crosslinked siRNA (e.g., contains a single crosslink),is modified at a 3′ terminus, contains a biotin at a 3′ terminus,contains a photocleavable biotin having the structure depicted in FIG. 8at a 3′ terminus, or contains a peptide (e.g., a Tat peptide),nanoparticle, peptidomimetic, organic molecule (e.g., a fluorescentdye), or dendrimer at a 3′ terminus. In some embodiments of the method,the siRNA derivative inhibits expression of the target gene at least30%. The cell can be a mammalian cell (e.g., human cell). In some cases,the concentration of the siRNA derivative administered to the cell orwithin the cell does not completely inhibit expression of the targetgene. In some embodiments, the modified siRNA is carried out in theabsence of a transfection reagent.

The invention includes a novel photocleavable biotin of the formuladepicted in FIG. 20, and the method of synthesizing the compound.

Exemplary siRNAs to be modified according to the methodologies describedherein are siRNAs targeting transcription elongation factors (TEFs), inparticular, DSIF and P-TEFb, as well as siRNAs targeting subunits ofsaid TEFs, in particular, CycT1, CDK9 and Spt5. siRNAs targeting TEFsare described in detail herein and in PCT/US03/24610. All combinationsof modifications described herein and siRNAs (and other RNAi agents)described, for example, in PCT/US03/24610, are the intended scope of theinstant patent application. Methods as described herein and, forexample, in PCT/US03/24610, featuring modified siRNAs (or RNAi agents)as described herein are further the intended scope of the instant patentapplication.

So that the invention may be more readily understood, certain terms arefirst defined.

The term “nucleoside” refers to a molecule having a purine or pyrimidinebase covalently linked to a ribose or deoxyribose sugar. Exemplarynucleosides include adenosine, guanosine, cytidine, uridine andthymidine. The term “nucleotide” refers to a nucleoside having one ormore phosphate groups joined in ester linkages to the sugar moiety.Exemplary nucleotides include nucleoside monophosphates, diphosphatesand triphosphates. The terms “polynucleotide” and “nucleic acidmolecule” are used interchangeably herein and refer to a polymer ofnucleotides joined together by a phosphodiester linkage between 5′ and3′ carbon atoms.

The term “RNA” or “RNA molecule” or “ribonucleic acid molecule” refersto a polymer of ribonucleotides. The term “DNA” or “DNA molecule” ordeoxyribonucleic acid molecule” refers to a polymer ofdeoxyribonucleotides. DNA and RNA can be synthesized naturally (e.g., byDNA replication or transcription of DNA, respectively). RNA can bepost-transcriptionally modified. DNA and RNA can also be chemicallysynthesized. DNA and RNA can be single-stranded (i.e., ssRNA and ssDNA,respectively) or multi-stranded (e.g., double stranded, i.e., dsRNA anddsDNA, respectively). “mRNA” or “messenger RNA” is single-stranded RNAthat specifies the amino acid sequence of one or more polypeptidechains. This information is translated during protein synthesis whenribosomes bind to the mRNA.

As used herein, the term “small interfering RNA” (“siRNA”) (alsoreferred to in the art as “short interfering RNAs”) refers to an RNA (orRNA analog) comprising between about 10-50 nucleotides (or nucleotideanalogs) which is capable of directing or mediating RNA interference.

The term “nucleotide analog”, also referred to herein as an “alterednucleotide” or “modified nucleotide” refers to a non-standardnucleotide, including non-naturally occurring ribonucleotides ordeoxyribonucleotides. Preferred nucleotide analogs are modified at anyposition so as to alter certain chemical properties of the nucleotideyet retain the ability of the nucleotide analog to perform its intendedfunction.

The term “oligonucleotide” refers to a short polymer of nucleotidesand/or nucleotide analogs. The term “RNA analog” refers to anpolynucleotide (e.g., a chemically synthesized polynucleotide) having atleast one altered or modified nucleotide as compared to a correspondingunaltered or unmodified RNA but retaining the same or similar nature orfunction as the corresponding unaltered or unmodified RNA. As discussedabove, the oligonucleotides may be linked with linkages which result ina lower rate of hydrolysis of the RNA analog as compared to an RNAmolecule with phosphodiester linkages. For example, the nucleotides ofthe analog may comprise methylenediol, ethylene diol, oxymethylthio,oxyethylthio, oxycarbonyloxy, phosphorodiamidate, phophoroamidate,and/or phosphorothioate linkages. Exemplary RNA analogues include sugar-and/or backbone-modified ribonucleotides and/or deoxyribonucleotides.Such alterations or modifications can further include addition ofnon-nucleotide material, such as to the end(s) of the RNA or internally(at one or more nucleotides of the RNA). An RNA analog need only besufficiently similar to natural RNA that it has the ability to mediate(mediates) RNA interference.

As used herein, the term “RNA interference” (“RNAi”) refers to aselective intracellular degradation of RNA. RNAi occurs in cellsnaturally to remove foreign RNAs (e.g., viral RNAs). Natural RNAiproceeds via fragments cleaved from free dsRNA which direct thedegradative mechanism to other similar RNA sequences. Alternatively,RNAi can be initiated by the hand of man, for example, to silence theexpression of target genes.

A siRNA having a “sequence sufficiently complementary to a target mRNAsequence to direct target-specific RNA interference (RNAi)” means thatthe siRNA has a sequence sufficient to trigger the destruction of thetarget mRNA by the RNAi machinery or process.

The term “cleavage site” refers to the residues, e.g. nucleotides, atwhich RISC* cleaves the target RNA, e.g., near the center of thecomplementary portion of the target RNA, e.g., about 8-12 nucleotidesfrom the 5′ end of the complementary portion of the target RNA. The term“upstream of the cleavage site” refers to residues, e.g., nucleotides ornucleotide analogs, 5′ to the cleavage site. Upstream of the cleavagesite with reference to the antisense strand refers to residues, e.g.nucleotides or nucleotide analogs 5′ to the cleavage site in theantisense strand.

The term “downstream of the cleavage site” refers to residues, e.g.,nucleotides or nucleotide analogs, located 3′ to the cleavage site.Downstream of the cleavage site with reference to the antisense strandrefers to residues, e.g., nucleotides or nucleotide analogs, 3′ to thecleavage site in the antisense strand.

The term “mismatch” refers to a basepair consisting of noncomplementarybases, e.g. not normal complementary G:C, A:T or A:U base pairs.

The term “phosphorylated” means that at least one phosphate group isattached to a chemical (e.g., organic) compound. Phosphate groups can beattached, for example, to proteins or to sugar moieties via thefollowing reaction: free hydroxyl group+phosphate donor→phosphate esterlinkage. The term “5′ phosphorylated” is used to describe, for example,polynucleotides or oligonucleotides having a phosphate group attachedvia ester linkage to the C5 hydroxyl of the 5′ sugar (e.g., the 5′ribose or deoxyribose, or an analog of same). Mono-, di-, andtriphosphates are common. Also intended to be included within the scopeof the instant invention are phosphate group analogs which function inthe same or similar manner as the mono-, di-, or triphosphate groupsfound in nature (see e.g., exemplified analogs.)

As used herein, the term “isolated” molecule (e.g., isolated nucleicacid molecule) refers to molecules which are substantially free of othercellular material, or culture medium when produced by recombinanttechniques, or substantially free of chemical precursors or otherchemicals when chemically synthesized.

The term “in vitro” has its art recognized meaning, e.g., involvingpurified reagents or extracts, e.g., cell extracts. The term “in vivo”also has its art recognized meaning, e.g., involving living cells, e.g.,immortalized cells, primary cells, cell lines, and/or cells in anorganism.

A target gene is a gene targeted by a compound of the invention (e.g., asiRNA (targeted siRNA), candidate siRNA derivative, siRNA derivative,modified siRNA, etc.), e.g., for RNAi-mediated gene knockdown. Oneportion of an siRNA is complementary (e.g., fully complementary) to asection of the mRNA of the target gene.

A gene “involved” in a disorder includes a gene, the normal or aberrantexpression or function of which effects or causes a disease or disorderor at least one symptom of said disease or disorder

The phrase “examining the function of a gene in a cell or organism”refers to examining or studying the expression, activity, function orphenotype arising therefrom.

Various methodologies of the instant invention include step thatinvolves comparing a value, level, feature, characteristic, property,etc. to a “suitable control”, referred to interchangeably herein as an“appropriate control”. A “suitable control” or “appropriate control” isany control or standard familiar to one of ordinary skill in the artuseful for comparison purposes. In one embodiment, a “suitable control”or “appropriate control” is a value, level, feature, characteristic,property, etc. determined prior to performing an RNAi methodology, asdescribed herein. For example, a transcription rate, mRNA level,translation rate, protein level, biological activity, cellularcharacteristic or property, genotype, phenotype, etc. can be determinedprior to introducing a siRNA of the invention into a cell or organism.In another embodiment, a “suitable control” or “appropriate control” isa value, level, feature, characteristic, property, etc. determined in acell or organism, e.g., a control or normal cell or organism,exhibiting, for example, normal traits. In yet another embodiment, a“suitable control” or “appropriate control” is a predefined value,level, feature, characteristic, property, etc.

A cell or culture that has not been contacted with a modified siRNA oran siRNA derivative is a control cell or control culture. The controlcell or control culture generally contains one or more reporter genesthat are expressed or one or more endogenous genes of interest, e.g.,for RNAi-mediated knockdown. In some embodiments of the invention, thecontrol cell or control culture contains an siRNA targeted to a reportergene or to an endogenous gene of interest. In some cases, the controlcell or control culture contains an introduced control sequence such asan antisense strand corresponding to the antisense strand of an siRNA ormodified siRNA.

A test cell or test culture contains one or more reporter genes that areexpressed or one or more expressed endogenous genes of interest, e.g.,for RNAi-mediated gene knockdown, and also contains a modified siRNA orsiRNA derivative targeted to a reporter gene or to an endogenous gene ofinterest.

Various aspects of the invention are described in further detail in thefollowing subsections.

I. siRNA Molecules

The present invention features “small interfering RNA molecules” (“siRNAmolecules” or “siRNA”), methods of making said siRNA molecules andmethods (e.g., research and/or therapeutic methods) for using said siRNAmolecules. An siRNA molecule of the invention is a duplex consisting ofa sense strand and complementary antisense strand, the antisense strandhaving sufficient complementarity to a target mRNA to mediate RNAi.Preferably, the strands are aligned such that there are at least 1, 2,or 3 bases at the end of the strands which do not align (i.e., for whichno complementary bases occur in the opposing strand) such that anoverhang of 1, 2 or 3 residues occurs at one or both ends of the duplexwhen strands are annealed. Preferably, the siRNA molecule has a lengthfrom about 10-50 or more nucleotides, i.e., each strand comprises 10-50nucleotides (or nucleotide analogs). More preferably, the siRNA moleculehas a length from about 15-45 nucleotides. Even more preferably, thesiRNA molecule has a length from about 18-25 nucleotides. The siRNAmolecules of the invention further have a sequence that is “sufficientlycomplementary” to a target mRNA sequence to direct target-specific RNAinterference (RNAi), as defined herein, i.e., the siRNA has a sequencesufficient to trigger the destruction of the target mRNA by the RNAimachinery or process.

The target RNA cleavage reaction guided by siRNAs (e.g., by siRNAs) ishighly sequence specific. In general, siRNA containing a nucleotidesequences identical to a portion of the target gene are preferred forinhibition. However, 100% sequence identity between the siRNA and thetarget gene is not required to practice the present invention. Thus theinvention has the advantage of being able to tolerate sequencevariations that might be expected due to genetic mutation, strainpolymorphism, or evolutionary divergence. For example, siRNA sequenceswith insertions, deletions, and single point mutations relative to thetarget sequence have also been found to be effective for inhibition.Moreover, not all positions of a siRNA contribute equally to targetrecognition. Mismatches in the center of the siRNA are most critical andessentially abolish target RNA cleavage. Mismatches upstream of thecenter or upstream of the cleavage site referencing the antisense strandare tolerated but significantly reduce target RNA cleavage. Mismatchesdownstream of the center or cleavage site referencing the antisensestrand, preferably located near the 3′ end of the antisense strand, e.g.1, 2, 3, 4, 5 or 6 nucleotides from the 3′ end of the antisense strand,are tolerated and reduce target RNA cleavage only slightly.

Sequence identity may determined by sequence comparison and alignmentalgorithms known in the art. To determine the percent identity of twonucleic acid sequences (or of two amino acid sequences), the sequencesare aligned for optimal comparison purposes (e.g., gaps can beintroduced in the first sequence or second sequence for optimalalignment). The nucleotides (or amino acid residues) at correspondingnucleotide (or amino acid) positions are then compared. When a positionin the first sequence is occupied by the same residue as thecorresponding position in the second sequence, then the molecules areidentical at that position. The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences (i.e., % homology=# of identical positions/total # ofpositions×100), optionally penalizing the score for the number of gapsintroduced and/or length of gaps introduced.

The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. In one embodiment, the alignment generated over a certainportion of the sequence aligned having sufficient identity but not overportions having low degree of identity (i.e., a local alignment). Apreferred, non-limiting example of a local alignment algorithm utilizedfor the comparison of sequences is the algorithm of Karlin and Altschul(1990) Proc. Natl. Acad. Sci. USA 87:2264-68, modified as in Karlin andAltschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-77. Such an algorithmis incorporated into the BLAST programs (version 2.0) of Altschul, etal. (1990) J. Mol. Biol. 215:403-10.

In another embodiment, the alignment is optimized by introducingappropriate gaps and percent identity is determined over the length ofthe aligned sequences (i.e., a gapped alignment). To obtain gappedalignments for comparison purposes, Gapped BLAST can be utilized asdescribed in Altschul et al., (1997) Nucleic Acids Res.25(17):3389-3402. In another embodiment, the alignment is optimized byintroducing appropriate gaps and percent identity is determined over theentire length of the sequences aligned (i.e., a global alignment). Apreferred, non-limiting example of a mathematical algorithm utilized forthe global comparison of sequences is the algorithm of Myers and Miller,CABIOS (1989). Such an algorithm is incorporated into the ALIGN program(version 2.0) which is part of the GCG sequence alignment softwarepackage. When utilizing the ALIGN program for comparing amino acidsequences, a PAM120 weight residue table, a gap length penalty of 12,and a gap penalty of 4 can be used.

Greater than 90% sequence identity, e.g., 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or even 100% sequence identity, between the siRNA and theportion of the target gene is preferred. Alternatively, the siRNA may bedefined functionally as a nucleotide sequence (or oligonucleotidesequence) that is capable of hybridizing with a portion of the targetgene transcript (e.g., 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50°C. or 70° C. hybridization for 12-16 hours; followed by washing).Additional preferred hybridization conditions include hybridization at70° C. in 1×SSC or 50° C. in 1×SSC, 50% formamide followed by washing at70° C. in 0.3×SSC or hybridization at 70° C. in 4×SSC or 50° C. in4×SSC, 50% formamide followed by washing at 67° C. in 1×SSC. Thehybridization temperature for hybrids anticipated to be less than 50base pairs in length should be 5-10° C. less than the meltingtemperature (Tm) of the hybrid, where Tm is determined according to thefollowing equations. For hybrids less than 18 base pairs in length, Tm(° C.)=2(# of A+T bases)+4(# of G+C bases). For hybrids between 18 and49 base pairs in length, Tm (° C.)=81.5+16.6(log 10[Na+])+0.41(%G+C)−(600/N), where N is the number of bases in the hybrid, and [Na+] isthe concentration of sodium ions in the hybridization buffer ([Na+] for1×SSC=0.165 M). Additional examples of stringency conditions forpolynucleotide hybridization are provided in Sambrook, J., E. F.Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., chapters9 and 11, and Current Protocols in Molecular Biology, 1995, F. M.Ausubel et al., eds., John Wiley & Sons, Inc., sections 2.10 and6.3-6.4, incorporated herein by reference. The length of the identicalnucleotide sequences may be at least about 10, 12, 15, 17, 20, 22, 25,27, 30, 32, 35, 37, 40, 42, 45, 47 or 50 bases.

In a preferred aspect, the invention features small interfering RNAs(siRNAs) that include a sense strand and an antisense strand, whereinthe antisense strand has a sequence sufficiently complementary to atarget mRNA sequence to direct target-specific RNA interference (RNAi)and wherein the sense strand and/or antisense strand is modified by thesubstitution of internal nucleotides with modified nucleotides, suchthat in vivo stability is enhanced as compared to a correspondingunmodified siRNA. As defined herein, an “internal” nucleotide is oneoccurring at any position other than the 5′ end or 3′ end of nucleicacid molecule, polynucleotide or oligonucleoitde. An internal nucleotidecan be within a single-stranded molecule or within a strand of a duplexor double-stranded molecule. In one embodiment, the sense strand and/orantisense strand is modified by the substitution of at least oneinternal nucleotide. In another embodiment, the sense strand and/orantisense strand is modified by the substitution of at least 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25 or more internal nucleotides. In another embodiment, the sense strandand/or antisense strand is modified by the substitution of at least 5%,10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95% or more of the internal nucleotides. In yet anotherembodiment, the sense strand and/or antisense strand is modified by thesubstitution of all of the internal nucleotides.

In yet another embodiment, the modified nucleotides are present only inthe antisense strand. In yet another embodiment, the modifiednucleotides are present only in the sense strand. In yet otherembodiments, the modified nucleotides are present in both the sense andantisense strand.

Preferred modified nucleotides or nucleotide analogues include sugar-and/or backbone-modified ribonucleotides (i.e., include modifications tothe phosphate-sugar backbone). For example, the phosphodiester linkagesof natural RNA may be modified to include at least one of a nitrogen orsulfur heteroatom. In preferred backbone-modified ribonucleotides thephosphoester group connecting to adjacent ribonucleotides is replaced bya modified group, e.g., of phosphothioate group. In preferredsugar-modified ribonucleotides, the 2′ moiety is a group selected fromH, OR, R, halo, SH, SR, NH₂, NHR, NR₂ or ON, wherein R is C₁-C₆ alkyl,alkenyl or alkynyl and halo is F, Cl, Br or I.

Preferred are 2′-fluro, 2′-amino and/or 2′-thio modifications.Particularly preferred modifications include 2′-fluoro-cytidine,2′-fluoro-uridine, 2′-fluoro-adenosine, 2′-fluoro-guanosine,2′-amino-cytidine, 2′-amino-uridine, 2′-amino-adenosine,2′-amino-guanosine, 2,6-diaminopurine, 4-thio-uridine; and/or5-amino-allyl-uridine. Additional exemplary modifications include5-bromo-uridine, 5-iodo-uridine, 5-methyl-cytidine, ribo-thymidine,2-aminopurine, 2′-amino-butyryl-pyrene-uridine, 5-fluoro-cytidine, and5-fluoro-uridine. 2′-deoxy-nucleotides can be used within modifiedsiRNAs of the instant invention, but are preferably included within thesense strand of the siRNA duplex. 2′-OMe nucleotides are less preferred.Additional modified residues have been described in the art and arecommercially available but are less preferred for use in the modifiedsiRNAs of the instant invention including, deoxy-abasic, inosine,N3-methyl-uridine, N6, N6-dimethyl-adenosine, pseudouridine, purineribonucleoside and ribavirin. Modification of the linkage betweennucleotides or nucleotide analogs is also preferred, e.g., substitutionof phosphorothioate linkages for phosphodiester linkages.

Also possible are nucleobase-modified ribonucleotides, i.e.,ribonucleotides, containing at least one non-naturally occurringnucleobase instead of a naturally occurring nucleobase. Bases may bemodified to block the activity of adenosine deaminase. Exemplarymodified nucleobases include, but are not limited to, uridine and/orcytidine modified at the 5-position, e.g., 5-(2-amino)propyl uridine,5-bromo uridine; adenosine and/or guanosines modified at the 8 position,e.g., 8-bromo guanosine; deaza nucleotides, e.g., 7-deaza-adenosine; O-and N-alkylated nucleotides, e.g., N6-methyl adenosine are suitable.

It should be noted that all modifications described herein may becombined. In a preferred embodiment, 2′-fluoro modified ribonucleotidesand 2′-deoxy ribonucleotides are combined and both are present withinthe antisense strand.

Preferably, an siRNA molecule of the invention will have athree-dimensional structure resembling A-form RNA helix. Morepreferably, an siRNA molecule of the invention will have an antisensestrand which is capable of adopting an A-form helix when in associationwith a target RNA (e.g., an mRNA). For this reason, 2′-fluro-modifiednucleotides are preferred, as siRNA made with such modified nucleotidesadopts an A-form helix confirmation. In particular, it is important thatan siRNA be capable of adopting an A-form helix in the portioncomplementary to the target cleavage site as it has been discovered thatthe major groove formed by the A-form helix at the cleavage site, andnot the RNA itself, is an essential determinant of RNAi. Morepreferably, a siRNA molecule will have exhibit increased cellular uptakewhen contacted with a cell, e.g., a human cell, as compared to anunmodified siRNA molecule. Even more preferably, a siRNA molecule willexhibit increased stability (i.e, resistance to cellular nucleases) ascompared to an unmodified siRNA molecule.

II. siRNA Derivatives

Discoveries have been made that elucidate certain mechanisms of RNAi.These discoveries indicate that the status of the 5′ hydroxyl terminusof the antisense strand of an siRNA determines RNAi activity, whereas a3′ terminus block is well tolerated in living cells. Furthermore,isolation of siRNA from human cells has revealed that 5′ hydroxyltermini of the antisense strands are phosphorylated. It has also beendiscovered that biotin, chemically linked to the 3′ terminus of an siRNA(e.g., a type of siRNA derivative), is not efficiently removed and thatsiRNAs having such 3′ biotins are effective in RNAi. In addition, it hasbeen found that there is no requirement for a perfect A-form helix insiRNA for interference effects, but an A-form structure is required forantisense-target RNA duplexes. Strikingly, crosslinking of the siRNAduplex by psoralen does not completely block RNA interference,indicating that complete unwinding of the siRNA helix is not necessaryfor RNAi activity in vivo. These results highlight the importance of 5′hydroxyl in the antisense strand of siRNA, which is essential toinitiate the RNAi pathway. Contrary to current beliefs, these data showthat RNA amplification by RNA-dependent RNA polymerase is not essentialfor RNAi in mammalian (e.g., human) cells.

Based on these discoveries, the invention includes modifications tosiRNA to create corresponding siRNA derivatives. The siRNA to bemodified can be naturally occurring or synthetic. Modifications includealtering a 3′ OH end of an siRNA to create a corresponding siRNAderivative with a new property such as increased stability or a label.In some embodiments, siRNA is modified by crosslinking between one ormore pairs of nucleotides in an siRNA, thereby creating another type ofsiRNA derivative. The invention also includes a novel photocleavablebiotin that is, for example, useful for labeling a 3′ OH terminus of ansiRNA.

In some aspects the invention relates to siRNA derivatives. An siRNAderivative is a double-stranded RNA-based structure that is 15-30nucleotides in length (e.g., 15-25 or in some cases, 21-25 nucleotidesin length), has certain features in common with a corresponding siRNA(an siRNA targeted to the same sequence as the siRNA derivative) such asthe ability to inhibit expression of a target sequence. The sequence ofthe antisense strand of an siRNA or an siRNA derivative is exactlycomplementary to at least a portion of the target mRNA. An siRNAtypically has a 2-3 nucleotide 3′ overhanging end, a 5′ phosphate (uponextraction from a cell) and a 3′ hydroxyl terminus. In addition, ansiRNA derivative has at least one of the following which is not afeature of siRNA: a label at the 3′ terminus (e.g., biotin or afluorescent molecule, the 3′ terminus is blocked, the 3′ terminus has acovalently linked group or compound (e.g., a nanoparticle or a peptide),the siRNA derivative does not form a perfect A-form helix, but theantisense strand of the siRNA derivative duplex does form an A-formhelix with target RNA, or the siRNA derivative is crosslinked (e.g., bypsoralen). Methods of synthesizing RNAs and modifying RNAs are known inthe art (e.g., Hwang et al., 1999, Proc. Nat. Acad. Sci. USA96:12997-13002; and Huq and Rana, 1997, Biochem. 36:12592-12599).

In some embodiments of the invention, an siRNA derivative also exhibitsa relatively low level of toxicity. For example, a concentration of ansiRNA derivative that inhibits expression of a targeted sequence hasrelatively low toxicity when at least 50% of the cells in a culturetreated with the siRNA derivative are viable when expression of thetargeted sequence is decreased by 50% compared to expression in a cellthat is not treated with the siRNA derivative. Low toxicity may beassociated with greater cell viability, e.g., at least 60%, 75%, 85%,90%, 95%, or 100%. Methods of measuring cell viability are known in theart and include trypan blue exclusion.

RNAi provides a new approach for elucidation of gene function and forinhibiting expression of undesirable genes, which is also known as “geneknockdown.” RNAi-mediated gene knockdown is useful for, e.g.,genome-wide analysis of gene function, target validation of potentiallytherapeutic genes, and therapies based on the elimination, reduction, orelimination of expression of a specific gene product. In addition,siRNAs are useful tools for cell biologists studying mammalian genefunction. For example, siRNAs are useful for the analysis of generalcell biological mechanisms such as mitosis, processing and traffickingof RNA transcripts, the formation of cellular junctions, and membranetrafficking. Reagents that can be used for such analyses (e.g., modifiedsiRNAs with increased stability or functional groups that endow an siRNAwith additional properties) have commercial value for use in suchresearch.

The invention provides siRNAs that have been chemically modified.Certain modifications confer useful properties to siRNA. For example,increased stability compared to an unmodified siRNA or a label that canbe used, e.g., to trace the siRNA, to purify an siRNA, or to purify thesiRNA and cellular components with which it is associated. Certainmodifications can also increase the uptake of the siRNA by a cell.

RNAi-mediated gene knockdown can cause a phenotype that is lethal ortoxic for a cell or the siRNA used to target a gene for knockdown mayaffect multiple pathways in the cell. Therefore, chemically modifiedsiRNAs (siRNA derivatives) that are less efficient than thecorresponding siRNA are still useful in some applications of RNAi. SiRNAderivatives containing certain functional groups such as biotin areuseful for affinity purification of proteins and molecular complexesinvolved in the RNAi mechanism. The invention also includes methods oftesting modified siRNAs for retention of the ability to act as an siRNA(e.g., in RNAi) and methods of using siRNA derivatives.

A. Crosslinked siRNA Derivatives

Some embodiments include the use of siRNAs that contain one or morecrosslinks between nucleic acids in the complementary strands of thesiRNA. Crosslinks can be introduced into an siRNA using methods known inthe art. In addition to crosslinking using psoralen (e.g., Example 1 andExample 9, infra; Wang et al., 1996, J. Biol. Chem. 271:16995-16998)other methods of crosslinking can be used. In some embodiments,photocrosslinks are made containing thiouracil (e.g., 4-thiouridine) orthioguanosine bases. In other embodiments, —SH linkers can be added tothe bases or sugar backbones, which are used to make S—S crosslinks. Insome cases, sugar backbones or amino groups at the C5 position of U, Ccan be labeled with benzophenone and other photo crosslinkers or withchemical crosslinkers. Methods of making such crosslinks are known inthe art (e.g., Wang and Rana, 1998, Biochem. 37:4235-4243; BioMosaics,Inc., Burlington, Vt.). In general, the stability in a cell or acell-free system of a crosslinked siRNA derivative is greater than thatof the corresponding siRNA. In some cases, the crosslinked siRNAderivative has less activity than the corresponding siRNA. The abilityof a crosslinked siRNA to inhibit expression of a target sequence can beassayed using methods known in the art for testing the activity of ansiRNA or by methods disclosed herein such as a dual fluorescencereporter gene assay.

In general, an siRNA derivative that is crosslinked contains onecrosslink between two nucleotides of a dsRNA sequence. In someembodiments, there are two or more crosslinks. Crosslinks are generallylocated near the 3′ terminus of the antisense strand, e.g., within about10 nucleotides of the 3′ terminus of the antisense strand, and generallywithin about 2-7 nucleotides of the 3′ terminus of the antisense strand.A crosslink is to be distinguished from ligation that joins the ends ofthe two strands of an siRNA. A mixture of crosslinked siRNA derivativesthat contains some molecules crosslinked at loci near the middle of thesiRNA or near the 5′ terminus of the antisense strand can also beuseful. Such mixtures can have less activity than a mixture of siRNAderivative that is crosslinked exclusively near the 3′ terminus, butretain sufficient activity to affect expression of a targeted sequence.

B. 3′ Modifications of siRNA

It has been discovered that the 3′ terminus of siRNA is not critical foractivity in RNAi. Therefore, modifications can be made to an siRNA tocreate an siRNA derivative. For example, molecules that are used foraffinity purification or as detectable tags can be covalently linked tothe 3′ terminus of an RNAi to create an siRNA derivative. Such RNAiderivatives are useful, e.g., for assaying an siRNA by transfecting acell with an siRNA derivative of the siRNA containing a detectable tagat the 3′ end and detecting the tag using methods known in the art.Examples of such tags that can be used for detection or affinitypurification of derivative siRNAs include biotin.

Methods that can be used to modify an siRNA are known in the art. Forexample, crosslinkers can be attached using amino-allyl couplingmethods, e.g., isothiocyanate, N-hydroxysuccinimide (NHS) esters(Amersham Biosciences Corp., Piscataway, N.J.). A number of differenttypes of molecules can be attached to a 3′ terminus using such methodsincluding dyes (e.g., Dyomics, Germany; Integrated DNA Technologies,Coralville, Iowa, ATTO-TEC, Siegen, Germany), dendrimers (e.g.,Dendritech, Midland, Mich.), and nanoparticles. Crosslinkers can beattached to amino-allyl uridine or amino groups at sugars using similarchemistry.

The invention includes conjugation of compounds to an siRNA. Primaryamines are the principal targets for NHS esters. For example, NHS estersof biotin can be conjugated to free amino groups at the 3′-end of ansiRNA duplex as described in the Examples.

In some embodiments, photocrosslinkers (e.g., thiouracil, thioguanosine,psoralens, benzophenones) are attached at 3′ terminus of an siRNA tocreate an siRNA derivative. Methods of synthesizing such modificationsare known in the art. Such an siRNA derivative can be crosslinked to thetarget cellular machinery in vitro and in vivo.

Other heterofunctional linkers can be used to modify the 3′ termini ofsiRNAs, for example, to link a peptide or a peptidomimetic oligomer toan siRNA (e.g., Tamilarasu et al., 2001, Bioorganic & MedicinalChemistry Letters 11:505-507). For example, one end of the pair to belinked (siRNA and peptide) can be made amine reactive and the otherthiol reactive. SiRNA that has been modified in this fashion can bedeprotected and linked to structures that, e.g., improve cellular uptakeof the resulting siRNA derivative compared to uptake of thecorresponding siRNA, are useful for tracing the siRNA derivative in thecell, or improve the stability of the siRNA derivative compared to thecorresponding siRNA. Example 18 illustrates the use of such amodification in which a deprotected and purified modified siRNA waslinked to Tat peptides, thereby improving cellular uptake of the siRNA.Such methods of attaching peptides, including Tat peptides, are known inthe art (e.g., Wang et al., 2001, Biochemistry 40:6458-6464). Methods ofsynthesizing peptides and peptidomimetics are known in the art and cangenerally be obtained from commercial sources (e.g., AnaSpec, San Jose,Calif.).

In another embodiment, the 3′ terminus of siRNA is labeled withdendrimer and/or nanoparticle structures that can enhance cellulartargeting activities without causing any known toxic effects. Inaddition, certain dendrimers are useful for facilitating uptake ofmolecules into cells, thus covalent linkage of such a dendrimer to the3′ terminus of an siRNA can increase the efficiency of uptake into acell of the resulting dendrimer siRNA derivative.

In other embodiments, a dyes can be linked to 3′ termini of an siRNA.Such dyes include those that are useful for energy transfer andfunctional assays, e.g., of helicase activity. For example, afluorescent donor dye such as isothiocyanate-fluorescein can be attachedto the 3′ end of the antisense strand of an siRNA. An acceptor dye(e.g., isothiocyanate rhodamine) can be attached to the 5′ end.RNA-containing amino groups at the 3′ or 5′ end can be obtained fromcommercial sources or appropriate dyes can be purchased and themolecules synthesized (Integrated DNA Technologies, Coralville, Iowa).Such a modified siRNA can be incubated with RISC complex that containshelicase. Fluorescence resonance energy transfer (FRET) signals will bealtered when the RNA helix of the modified siRNA is unwound.

Modification of the 3′ end can also include attachment of photocleavablecompounds such as biotin. This is illustrated in Example 19. RNAiderivatives with photocleavable compounds attached to the 3′ terminusare useful, e.g., for isolating proteins and other molecular complexesthat bind to an siRNA. For example, photocleavable biotin can beattached to an siRNA. The resulting derivative is incubated with a celllysate or transfected into cells. After a suitable incubation time, thebiotin siRNA derivative is retrieved using avidin attached to asubstrate (e.g., beads). After washing, the biotin is photocleaved fromthe siRNA, thus releasing the siRNA and its interacting proteins. Theseproteins can then be subjected to further analysis using methods knownin the art.

C. Photocleavable Biotin

The invention includes a method of synthesizing a novel photocleavablebiotin that is depicted in FIG. 8. The novel photocleavable biotin isuseful for methods in which photocleavable biotins are presently usedsuch as the biotin pull out assay described in Examples 1 and 5. Theadvantage of this novel photocleavable biotin is its increasedsensitivity compared to other photocleavable biotins that are presentlyknown and commercially available. The advantages of the newphotocleavable biotin disclosed herein include the following features ofhaving a photolabile linker that is more efficiently cleaved, thecompound contains a longer chain between the biotin and photolabilearomatic ring, and it makes an amide link with the target protein orother compound of interest. The novel photocleavable compound is anoxygenated nitrobenzyl system (in contrast to compounds having only anitrobenzyl system) and cleaves efficiently when irradiated at 360 nm(J. Org. Chem., 1995, 60, 7328-7333; Burgess et al., 1997, J. Org. Chem.62:5662-5663).

The synthesis of probe 6 (novel photocleavable biotin) consists of sixreaction steps, which are depicted in the following scheme.

Synthesis of the amine 3: To prepare amine 3, a stirred solution of 0.50g (0.96 mmol) of the photo-linker 1 in 5 ml of anhydrous DMF, was added0.15 ml (0.96 mmol) of diisopropylcarbodiimide and 0.13 g (0.96 mmol) ofHOBt. A solution of 0.18 g (0.96 mmol) of compound 2 was then added in 2ml of DMF dropwise. The reaction mixture was then stirred overnight atroom temperature. After the completion of the reaction (checked by TLCanalysis), the solvent was evaporated at reduced pressure. Flash columnchromatography on silica gel with 85:15=EtOAC:MeOH afforded 0.64 g(96.5%) of the pure product. To the product thus obtained, 25 ml of 1:1CH₂Cl₂:TFA was added and the mixture was stirred at room temperature for30 minutes. The solvent was evaporated and the crude product wasco-evaporated twice in anhydrous DMF. This material is used for furthercoupling reactions.

Synthesis of the Biotinylated Product 4:

To synthesize biotinylated product 4, a mixture of 0.54 g (0.91 mmol) ofcompound 3, 0.15 ml (0.91 mmol) of diisopropylcarbodiimide, and 0.13 g(0.91 mmol) of HOBt in 5 ml of 1:1 DMF:NMP, was added to a solution of0.23 g (0.91 mmol) of (+)-Biotin in 2 ml of NMP. The reaction mixturewas then stirred at room temperature for 10 hours. After the completionof the reaction (as analyzed by HPLC), the solvent was removed underreduced pressure. Excess reagents and impurities were then removed byprecipitating the product 4 in a mixture of 90:10 CH₂Cl₂:MeOH to afford0.69 g (92.5%) of the pure product.

Synthesis of the Acid 5:

To a stirred solution of 0.69 g (0.85 mmol) of 4 in 10 ml of anhydrousDMF, was added 0.17 ml (1.68 mmol) of piperidine. The reaction mixturewas stirred at room temperature for 3 hours and concentrated atrotavapor under reduced pressure.

Excess reagents and side products were then removed by adding CH₂Cl₂,while the biotinylated amine precipitated out. It was then collected byfiltering through a sintered funnel and vacuum dried.

To the amine thus obtained, was added 0.13 g (1.28 mmol) of succinicanhydride in 5 ml of DMF and the reaction mixture was stirred at roomtemperature for 5 hours. It was then concentrated and the product 5 wasprecipitated by CH₂Cl₂ to afford 0.48 g (82%) of the pure (HPLC pure)product.

Synthesis of the Succinimidyl Ester 6:

To a stirred solution of 95 mg (0.14 mmol) of 5, 22 μL (0.14 mmol) ofdiisopropylcarbodiimide and 20 mg (0.14 mmol) of HOBt in 2 ml ofanhydrous DMF, was added 16 mg (0.14 mmol) of N-hydroxysuccinimide in0.5 ml of DMF. The pH of the reaction mixture was brought up to between8 and 9 and it was stirred overnight at room temperature. Concentrationat reduced pressure and HPLC purification using a preparative columnafforded 90 mg (84%) of 6 in pure form.

This novel photocleavable biotin is useful, e.g., for labeling siRNA asdescribed herein.

D. Use of an siRNA Derivative for Affinity Purification of RNAiComponents

An siRNA derivative can be used to affinity purify proteins involved inRNAi and to determine characteristics of molecules that participate inRNAi. An siRNA derivative can be used for affinity purification of RNAiproteins from various organisms, e.g., worms (such as Caenorhabditiselegans), insects (such as Drosophila melanogaster), and mammals (e.g.,mice, rats, domestic animals, and humans). For example, an siRNAderivative that has been modified by the addition of a molecule at a 3′terminus that can be used for crosslinking the siRNA derivative to asolid substrate is useful for, e.g., recovering an siRNA containing sucha modification from a cell (see the biotin pull out assay in Examples 1and 5) or for isolating components of the RNAi machinery such as RISCthat bind to the siRNA derivative. Such molecules provide insight intothe mechanism of RNAi in mammalian cells and additional targets forcompounds that inhibit or enhance RNAi activity. Methods for attaching acompound to a substrate for use in purification methods and methods foraffinity purification of proteins are known in the art.

III. Efficacy Assays

The invention further features assaying compounds of the invention thathave been altered in at least one of the features described herein whoseefficacy for modulating expression of a target RNA is not established.In one embodiment, the invention features methods of assaying theability of a compound of the invention (e.g., a siRNA, candidate RNAiderivative, modified siRNA, etc.) to modulate (e.g., inhibit) expressionof a target RNA using a dual fluorescence system. Other assay systemsknown in the art that measure the efficacy of an siRNA can be used toevaluate whether a modified siRNA is an siRNA derivative. In general,the ability of an siRNA derivative to inhibit detectable expression of atarget RNA is at least 10%, 20%, or 30% compared to expression of thetarget in the absence of the RNAi derivative. In some cases, expressionof the target sequence is inhibited 50%, 75%, 85%, 90%, or 100%.

A compound of the invention (e.g., a siRNA, candidate RNAi derivative,modified siRNA, etc.) can be tested for its ability to inhibitexpression of a targeted gene. For example, candidate RNAi derivativesthat can inhibit such expression are identified as siRNA derivatives.Any system in which RNAi activity can be detected can be used to testthe activity of a compound of the invention (e.g., a siRNA, candidateRNAi derivative, modified siRNA, etc.). In general, a system in whichRNAi activity can be detected is incubated in the presence and absenceof a compound of the invention (e.g., a siRNA, candidate RNAiderivative, modified siRNA, etc.

The invention includes a dual fluorescence reporter gene assay (DFRGassay) that can be used to test a compound of the invention (e.g., asiRNA, candidate RNAi derivative, modified siRNA, etc.). The DFRG assaycan also be used, for example, to test the ability of these and othertypes of compounds to inhibit expression of a targeted gene (i.e., RNAiinhibitors).

In the DFRG assay, cells are used that have RNAi activity and contain atleast two reporter genes that encode and can express at least twodifferent fluorescent proteins. Alternatively, at least one of thereporter genes can encode hybrid proteins comprising a portion thatcorresponds to a reporter protein and a portion that corresponds to aprotein of interest (i.e., is translated from an mRNA that is targetedby the siRNA or modified siRNA used in the assay). The fluorescenceemission spectra of the two proteins are such that they can bedistinguished when expressed simultaneously, e.g., red fluorescentprotein (RFP) and green fluorescent protein (GFP). One reporter gene isused as a reference. The reporter cell is transfected with a compound ofthe invention (e.g., a siRNA, candidate RNAi derivative, modified siRNA,etc.), for example, an siRNA that has been chemically modified at 3′terminus, contains at least one crosslink between the two strands of thesiRNA, or both. The compound of the invention (e.g., a siRNA, candidateRNAi derivative, modified siRNA, etc.) is targeted to one of thereporter gene sequences. In some cases, the cell is co-transfected withthe reporter genes and the compound of the invention (e.g., a siRNA,candidate RNAi derivative, modified siRNA, etc.). The cell is incubatedfor a time sufficient to produce detectable reporter proteins in theabsence of the compound of the invention (e.g., a siRNA, candidate RNAiderivative, modified siRNA, etc.). After incubation, the level offluorescence is measured using methods known in the art. Generally,after incubation, the cell is lysed and the lysate is cleared andprotein concentration determined. An aliquot of the lysate is thenassayed for fluorescence intensity.

The ratio of fluorescence emission intensities between the two reportergenes is compared to a control to standardize the ratio. Normalizedratios of less than one (i.e., less fluorophore expression in the cellcontacted with the compound of the invention (e.g., a siRNA, candidateRNAi derivative, modified siRNA, etc.) than in the control cell)indicate target sequence-specific interference.

In one embodiment, the invention includes a method of determiningwhether a candidate siRNA derivative is an siRNA derivative. The methodincludes the steps of obtaining a reporter cell comprising two differentfluorescent reporter genes, transfecting the reporter cell with acandidate siRNA derivative targeted to one of the fluorescent reportergenes, thus creating a test cell; incubating the test cell for a timesufficient for a reporter cell to express detectable levels of thefluorescent reporter proteins encoded by the fluorescent reporter genes;determining the fluorescence intensity of each fluorescent reporterprotein in the test cell; and determining the ratio of the level offluorescence intensity between the two fluorescent reporter proteins inthe test cell and normalizing the ratio to the ratio of fluorescenceintensity in a control reporter cell that was not transfected with thecandidate siRNA derivative, such that a normalized ratio of less thanone indicates that the candidate siRNA derivative is an siRNAderivative. In some embodiments of this method, the control reportercell is transfected with an antisense sequence that is complementary tothe targeted reporter gene. In some embodiments, the candidate siRNAderivative is a crosslinked siRNA (e.g., the modified siRNA contains asingle crosslink), the candidate siRNA derivative is psoralencrosslinked, the candidate siRNA derivative is modified at a 3′ terminus(e.g., the modified siRNA comprises a biotin at a 3′ terminus), or themodified siRNA contains a photocleavable biotin having the structuredepicted in FIG. 20 at a 3′ terminus. The candidate siRNA derivative cancontain a peptide (e.g., a Tat peptide), nanoparticle, peptidomimetic,organic molecule (e.g., a fluorescent dye) or dendrimer at a 3′terminus. In some cases, the two reporter proteins are Green FluorescentProtein (GFP) and Red Fluorescent Protein (RFP). In some cases, thenormalized ratio is at least 0.3.

The control ratio used for normalization is determined by transfecting acell with the two reporter genes, incubating, and determining the ratioof fluorescence intensities from the two cells as described above for atest cell. In some embodiments, the control cell is transfected with thereporter genes and with an antisense RNA that is specific for thereporter gene that is targeted by the compound of the invention (e.g., asiRNA, candidate RNAi derivative, modified siRNA, etc.). Methods ofdesigning and selecting siRNAs are known in the art. In some cases, thetargeted region in the mRNA and the sequence in the siRNA duplex arechosen using the following guidelines. The targeted sequence isgenerally selected from the open reading frame region from the cDNAsequence of the targeted gene. In general the target site is at least75-100 nucleotides downstream from the start codon. Neither the 5′ nor3′ untranslated regions and regions near the start codon are generallyused for targeting because these may be richer in regulatory proteinbinding sites. After locating the first AA dimer located about 100 basesdownstream from the start codon, the next 19 nucleotides following theAA dimer are recorded The percentage of guanosines and cytidines (G/Ccontent) of the AA-N19-21 base sequence is determined. The G/C contentof this short sequence must be less than 70% and greater than 30% foruse as siRNA. In general, the G/C content of the sequence is about 50%.If the selected sequence does not meet these criteria, the searchcontinues downstream to the next AA dimer until the G/C conditions aremet. To ensure that only one gene is targeted by the sequence, theselected sequence (generally about 21 nucleotides) is subjected to aBLAST search (NCBI database) against EST libraries.

In some embodiments of the invention, proteins from the lysates areprepared as described above and analyzed using Western blotting.Briefly, the proteins prepared from the transfected cells (control cellsand test cells) are subjected to SDS-PAGE (e.g., in a 10% gel) andtransferred to a membrane suitable for Western blotting (for example, aPVDF membrane). The membrane is immunoblotted using methods known in theart to detect the fluorescent reporter proteins. In general, a proteinthat can be used as a control for protein loading (such as ahousekeeping protein) is also detected. Less expression of the targetedprotein compared to control indicates that the test sequence (e.g.modified siRNA) is effective for target sequence-specific interference.

Cells to be used in a DFRG assay are generally cultured mammalian cells,e.g., human cells. The cells can be immortal, primary, or secondarycells. Cells from other organisms that exhibit RNAi or RNAi-typeactivity such as quelling can also be used. Such cells include thosefrom fungi, plants, invertebrates (e.g., Drosophila melanogaster andCaenorhabditis elegans), and vertebrates (e.g., zebrafish and mouse).Fluorescent molecules that can be used in DFRG assays are pairs offluorescent molecules whose emission spectra can be distinguished whenthere is simultaneous emission. Examples of such pairs include GreenFluorescent Protein (GFP) and Red Fluorescent Protein (RFP). Additionalexamples can be selected, e.g., from those shown in Table 1.

TABLE I LIVING COLORS FLUORESCENT PROTEINS Excit./Emiss. ExtinctionFluor. Maxima Coef- Quantum Protein (nm) ficient Yield Reference DsRed558/583 22,600 0.23 Matz et al., 1999 EGFP 488/507 56,000 0.60 D. W.Piston, EYFP 513/527 84,000 0.61 {close oversize bracket} VanderbiltECFP 433/475 28,000 0.40 University, EBFP 380/440 31,000 0.18 Personalcomm.

IV. Production

RNA may be produced enzymatically or by partial/total organic synthesis,any modified ribonucleotide can be introduced by in vitro enzymatic ororganic synthesis. In one embodiment, a siRNA is prepared chemically.Methods of synthesizing RNA molecules are known in the art, inparticular, the chemical synthesis methods as de scribed in Verma andEckstein (1998) Annul Rev. Biochem. 67:99-134. In another embodiment, asiRNA is prepared enzymatically. For example, a ds-siRNA can be preparedby enzymatic processing of a long ds RNA having sufficientcomplementarity to the desired target mRNA. Processing of long ds RNAcan be accomplished in vitro, for example, using appropriate cellularlysates and ds-siRNAs can be subsequently purified by gelelectrophoresis or gel filtration. ds-siRNA can then be denaturedaccording to art-recognized methodologies. In an exemplary embodiment,RNA can be purified from a mixture by extraction with a solvent orresin, precipitation, electrophoresis, chromatography, or a combinationthereof. Alternatively, the RNA may be used with no or a minimum ofpurification to avoid losses due to sample processing. Alternatively,the single-stranded RNAs can also be prepared by enzymatic transcriptionfrom synthetic DNA templates or from DNA plasmids isolated fromrecombinant bacteria. Typically, phage RNA polymerases are used such asT7, T3 or SP6 RNA polymerase (Milligan and Uhlenbeck (1989) MethodsEnzymol. 180:51-62). The RNA may be dried for storage or dissolved in anaqueous solution. The solution may contain buffers or salts to inhibitannealing, and/or promote stabilization of the single strands.

In one embodiment, siRNAs are synthesized either in vivo, in situ, or invitro. Endogenous RNA polymerase of the cell may mediate transcriptionin vivo or in situ, or cloned RNA polymerase can be used fortranscription in vivo or in vitro. For transcription from a transgene invivo or an expression construct, a regulatory region (e.g., promoter,enhancer, silencer, splice donor and acceptor, polyadenylation) may beused to transcribe the siRNA. Inhibition may be targeted by specifictranscription in an organ, tissue, or cell type; stimulation of anenvironmental condition (e.g., infection, stress, temperature, chemicalinducers); and/or engineering transcription at a developmental stage orage. A transgenic organism that expresses siRNA from a recombinantconstruct may be produced by introducing the construct into a zygote, anembryonic stem cell, or another multipotent cell derived from theappropriate organism.

V. Targets

In one embodiment, the target mRNA of the invention specifies the aminoacid sequence of a cellular protein (e.g., a nuclear, cytoplasmic,transmembrane, or membrane-associated protein). In another embodiment,the target mRNA of the invention specifies the amino acid sequence of anextracellular protein (e.g., an extracellular matrix protein or secretedprotein). As used herein, the phrase “specifies the amino acid sequence”of a protein means that the mRNA sequence is translated into the aminoacid sequence according to the rules of the genetic code. The followingclasses of proteins are listed for illustrative purposes: developmentalproteins (e.g., adhesion molecules, cyclin kinase inhibitors, Wnt familymembers, Pax family members, Winged helix family members, Hox familymembers, cytokines/lymphokines and their receptors,growth/differentiation factors and their receptors, neurotransmittersand their receptors); oncogene-encoded proteins (e.g., ABLI, BCLI, BCL2,BCL6, CBFA2, CBL, CSFIR, ERBA, ERBB, EBRB2, ETSI, ETSI, ETV6, FGR, FOS,FYN, HCR, HRAS, JUN, KRAS, LCK, LYN, MDM2, MLL, MYB, MYC, MYCLI, MYCN,NRAS, PIM I, PML, RET, SRC, TALI, TCL3, and YES); tumor suppressorproteins (e.g., APC, BRCA1, BRCA2, MADH4, MCC, NF I, NF2, RB I, TP53,and WTI); and enzymes (e.g., ACC synthases and oxidases, ACP desaturasesand hydroxylases, ADP-glucose pyrophorylases, ATPases, alcoholdehydrogenases, amylases, amyloglucosidases, catalases, cellulases,chalcone synthases, chitinases, cyclooxygenases, decarboxylases,dextriinases, DNA and RNA polymerases, galactosidases, glucanases,glucose oxidases, granule-bound starch synthases, GTPases, helicases,hernicellulases, integrases, inulinases, invertases, isomerases,kinases, lactases, lipases, lipoxygenases, lysozymes, nopalinesynthases, octopine synthases, pectinesterases, peroxidases,phosphatases, phospholipases, phosphorylases, phytases, plant growthregulator synthases, polygalacturonases, proteinases and peptidases,pullanases, recombinases, reverse transcriptases, RUBISCOs,topoisomerases, and xylanases).

In a preferred aspect of the invention, the target mRNA molecule of theinvention specifies the amino acid sequence of a protein associated witha pathological condition. For example, the protein may be apathogen-associated protein (e.g., a viral protein involved inimmunosuppression of the host, replication of the pathogen, transmissionof the pathogen, or maintenance of the infection), or a host proteinwhich facilitates entry of the pathogen into the host, drug metabolismby the pathogen or host, replication or integration of the pathogen'sgenome, establishment or spread of infection in the host, or assembly ofthe next generation of pathogen. Alternatively, the protein may be atumor-associated protein or an autoimmune disease-associated protein.

In one embodiment, the target mRNA molecule of the invention specifiesthe amino acid sequence of an endogenous protein (i.e., a proteinpresent in the genome of a cell or organism). In another embodiment, thetarget mRNA molecule of the invention specified the amino acid sequenceof a heterologous protein expressed in a recombinant cell or agenetically altered organism. In another embodiment, the target mRNAmolecule of the invention specified the amino acid sequence of a proteinencoded by a transgene (i.e., a gene construct inserted at an ectopicsite in the genome of the cell). In yet another embodiment, the targetmRNA molecule of the invention specifies the amino acid sequence of aprotein encoded by a pathogen genome which is capable of infecting acell or an organism from which the cell is derived.

By inhibiting the expression of such proteins, valuable informationregarding the function of said proteins and therapeutic benefits whichmay be obtained from said inhibition may be obtained.

VI. Targeting Transcription Elongation Factors

Positive transcription elongation factor complex b (P-TEFb), which iscomposed of two subunits, CDK9 and cyclin T1 (CycT1) (Garber et al.,Genes & Dev., 12:3512-3527 (1998)), allows the transition to productiveelongation, producing longer mRNA transcripts (Price (2000), supra). Twonegative transcription elongation factors, DSIF (DRBsensitivity-inducting factor; DRB is5,6-dichloro-1-β-D-ribofuranosylbenzimidazole) and NELF (negativeelongation factor), have been identified and characterized (Wada et al.,Genes Dev. 12:343-56 (1998); Yamaguchi et al., Cell 97:41-51 (1999)).DSIF is composed of at least two subunits, one 14-kDa and one 160-kDa,which are homologs of the Saccharomyces cerevisiae transcription factorsSpt5 and Spt4, respectively (Hartzog et al., Genes Dev. 12:357-369(1998)). NELF is composed of five polypeptides, named as NELF-A to -E,and contains a subunit identical to RD, a putatitive RNA-binding protein(containing arginine-aspartic acid (RD) dipeptide repeats) of unknownfunction. DSIF and NELF function cooperatively and strongly repress RNApol II elongation (Yamaguchi et al., supra). In the absence of P-TEFb,DSIF plays the role of a negative regulator in transcription (Wada etal., EMBO J. 17:7395-7403 (1998)). DSIF subunit Spt5 also has a positiveelongation activity in Tat transactivation (Wu-Baer et al., J. Mol.Biol. 277:179-197 (1998); Kim et al., Mol. Cell. Biol. 19:5960-598(1999)). Another transcription elongation factor, Spt6, has beenidentified which is functionally related to Spt5; Spt5 and Spt6 havebeen shown to colocalize at regions of active transcription as well asat certain stress response genes induced by heat shock (Kaplan et al.,Genes Dev. 14:2623-2634 (2000); Andrulis et al., Genes Dev. 14:2635-2649 (2000)).

Among the genes regulated in this manner are several protooncogenes(c-myc, c-myb, c-fos); c-fms, the gene encoding macrophage colonystimulating factor 1 (CSF-1) receptor; the gene encoding adenosinedeaminase; a collection of stress response genes including hsp70; andgenes involved in replication and pathogenesis of HIV-1 and HIV-2.

One elegant example of transcription elongation control is the mechanismof HIV-1 gene expression (reviewed in: Cullen 1998 Cell 93:685-92;Emerman and Malin 1998 Science 280:1880-4; Jeang et al. 1999 J Biol Chem274:28837-40; Jones 1997 Genes Dev 11:2593-2599; Karm 1999 J Mol Biol.293:235-254; Taube et al. 1999 Virology 264:245-253). The HIV-1transcriptional activation mechanism requires Tat interactions with thehuman Cyclin T1 (hCycT1) subunit of P-TEFb that recruits the kinasecomplex to the pol II elongation machinery (Bieniasz et al. 1998 EMBO J.17:7056-65; Herrmann and Rice 1995 J. Virol. 69:1612-1620; Herrmann andRice 1993 Virology 197:601-608; Isel and Karn 1999 J. Mol Biol.290:929-941; Jones 1997 Genes Dev. 11:2593-2599; Mancebo et al. 1997Genes Dev 11:2633-2644; Taube et al. 1999 Virology 264: 245-253; Wei etal. 1998 Cell 92:451-62; Yang et al. 1997 Proc Natl Acad Sci USA94:12331-12336; Zhu et al. 1997 Genes Dev. 11:2622-32). The pol II CTD,and Spt5 are also intimately connected to this regulation of HIV geneexpression by Tat and P-TEFb. During HIV transcription, P-TEFb, which isinitially found as a component of the pol II preinitiation complex(PIC), travels with the transcription elongation complex (TEC) as itmoves along the HIV transcription unit (Ping and Rana 1999 J Biol Chem274:7399-7404). In contrast, DSIF and NELF are not present in the PIC,but associate with the TEC at promoter proximal positions and thentravel with the TECs down the template (Ping and Rana 2001 J Biol Chem276:12951-12958).

Based, at least in part, on the findings presented in ExamplesXX-XXXIII, the present invention relates to methods of modulating (e.g.,decreasing) the activity of transcription elongation factors (TEFs) andmore specifically to ribonucleic acid interference (RNAi) of TEFs (e.g.,positive transcription elongation factors or P-TEFs) or subunits thereof(e.g., the P-TEFb subunits CDK9 and CycT1).

In one embodiment, RNA interference (RNAi) methods (e.g., featuringsiRNAs, siRNA derivative, a modified siRNA, etc., as described herein)are used to specifically silence one or more TEFs, e.g., P-TEFb, DSIFand/or Spt6. These RNAi methods can be used to reduce HIV infectivityand to regulate genes involved in cell proliferation anddifferentiation, e.g., genes that have been correlated with diseases anddisorders characterized by unwanted or aberrant cellular proliferationor differentiation, such as cancer. In one embodiment, the unwantedcellular proliferation is cancer, for instance, carcinomas, sarcomas,metastatic disorders, and hematopoietic neoplastic disorders.

In one embodiment, the target region of the mRNA sequence is locatedfrom 100 to 300 nucleotides downstream (3′) of the start of translationof the TEF mRNA. In another embodiment, the target region of the mRNAsequence is located in a 5′ untranslated region (UTR) or a 3′ UTR of themRNA of a TEF, e.g., CDK9, CycT1, Spt4, Spt5, or Spt6.

In another aspect, the invention features methods of treating a subjecthaving a disorder characterized by unwanted cellular proliferation,e.g., cancer, e.g., carcinomas, sarcomas, metastatic disorders andhematopoietic neoplastic disorders (e.g., leukemias), or proliferativeskin disorders, e.g., psoriasis, by administering to the subject anamount of a nucleic acid composition, e.g., a therapeutic composition,of the invention, effective to inhibit TEF activity. As used herein,inhibiting P-TEF activity refers to a reduction in the activity of TEF,e.g., by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%.

In another aspect, the invention provides a method of treating a subjectinfected with HIV by administering to the subject an amount of thenucleic acid compositions, e.g., the therapeutic compositions, of theinvention, effective to inhibit TEF expression or activity. In anotheraspect, the invention features a method of treating a subject having adisorder characterized by aberrant or unwanted expression of a genewhose expression is regulated by a TEF, e.g., CDK9, CycT1, Spt4, Spt5and/or Spt6, by administering to the subject an amount of the nucleicacid compositions, e.g., the therapeutic compositions, of the invention,effective to inhibit TEF expression or activity.

In another aspect, the invention features a method of treating a subjecthaving a disorder characterized by aberrant or unwanted expression oractivity of a TEF, e.g., CDK9, CycT1, Spt4, Spt5 and/or Spt6 byadministering to the subject an amount of the nucleic acid compositions,e.g., the therapeutic compositions, of the invention, effective toinhibit TEF expression or activity. In one embodiment, the disorder isHIV/AIDS. In another embodiment, the disorder is cancer, e.g.,carcinomas, sarcomas, metastatic disorders and hematopoietic neoplasticdisorders, e.g., leukemia.

1. TEF Nucleic Acid Targets

In one aspect, the invention features compositions (e.g., siRNAs, siRNAderivatives, modified siRNAs, etc.) that are targeted to a CDK9, CycT1,Spt4, Spt5, or Spt6 RNA.

The mRNA sequence of CDK9 can be any ortholog of CDK9, such as sequencessubstantially identical to the S. cerevisiae, human, C. elegans, D.melanogaster, or mouse CDK9, including but not limited to GenBankAccession Nos. NM_001261 (GI:17017983) (SEQ ID NO:2) (correspondingprotein sequence: NP_001252) (human); P50750 (human); NP_570930 (mouse);BA C40824 (mouse); NP_477226 (fruit fly); NP_492906 (C. elegans); orNP_492907 (C. elegans). The mRNA sequence of CycT1 can be any orthologof CycT1, such as sequences substantially identical to the S.cerevisiae, human, or mouse CycT1, including but not limited to GenBankAccession Nos. AF048730 (GI:2981195) (corresponding protein sequence:AAC39664) (human); NM_001240 (GI:17978465) (corresponding proteinsequence: NP_001231) (human); AAN73282 (chimpanzee); NP_033963 (mouse);AAD17205 (mouse); QDQWV9 (mouse); AAM74155 (goat); or AAM74156 (goat).

The mRNA sequence of Spt4 can be any ortholog of Spt4, such as sequencessubstantially identical to the S. cerevisiae, human, or mouse Spt4,including but not limited to GenBank Accession Nos. NM_003168(GI:4507310) (human Spt4); U38817 (GI:1401054) (humanSpt4); U38818(GI:1401052) (human Spt4); U43923 (GI:1297309)(human Spt4); NM_009296(GI:6678180) (mouse Spt4); U43154 (GI:1401065) (mouse Spt4) or M83672(S. cerevisiae Spt4). The mRNA sequence of Spt5 can be any ortholog ofSpt5, such as sequences substantially identical to the S. cerevisiae,human, or mouse Spt5, including but not limited to GenBank AccessionNos. BCO2403 (GI: 18848307) (human Spt5), NM 003169 (GI:20149523) (humanSpt5); AB000516 (GI:2723379) (human Spt5); AF 040253 (GI:4104823) (humanSpt5); U56402 (GI:1845266) (human Spt5); NM013676 (GI:22094122) (mouseSpt5); U888539 (mouse Spt5); or M 62882 (S. cerevisiae Spt5). The mRNAsequence of Spt6 can be any ortholog of Spt6, such as sequencessubstantially identical to the S. cerevisiae or mouse Spt6, includingbut not limited to NM_009297 (GI:6678182) (mouse Spt6) or M34391 (S.cerevisiae Spt6).

2. siRNA Molecules

The compositions (e.g., siRNAs, siRNA derivatives, modified siRNAs,etc.) of the invention include dsRNA molecules comprising 16-30, e.g.,16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30nucleotides in each strand, wherein one of the strands is substantiallyidentical, e.g., at least 80% (or more, e.g., 85%, 90%, 95%, or 100%)identical, e.g., having 3, 2, 1, or 0 mismatched nucleotide(s), to atarget region in the mRNA of CDK9, CycT1, Spt4, Spt5, or Spt6, and theother strand is identical or substantially identical to the firststrand. The compositions of the invention can be chemically synthesized,or can be transcribed in vitro from a DNA template, or in vivo from,e.g., shRNA. The dsRNA molecules can be designed using any method knownin the art, for instance, by using the following protocol:

-   -   A. Beginning with the AUG start codon, look for AA dinucleotide        sequences; each AA and the 3′ adjacent 16 or more nucleotides        are potential siRNA targets (see FIGS. 15, 16, 34, 35, 36).        siRNAs taken from the 5′ untranslated regions (UTRs) and regions        near the start codon (within about 75 bases or so) may be less        useful as they may be richer in regulatory protein binding        sites, and UTR-binding proteins and/or translation initiation        complexes may interfere with binding of the siRNP or RISC        endonuclease complex. Thus, in one embodiment, the nucleic acid        molecules are selected from a region of the cDNA sequence        beginning 50 to 100 nt downstream of the start codon. Further,        siRNAs with lower G/C content (35-55%) may be more active than        those with G/C content higher than 55%. Thus in one embodiment,        the invention includes nucleic acid molecules having 35-55% G/C        content. In addition, the strands of the siRNA can be paired in        such a way as to have a 3′ overhang of 1 to 4, e.g., 2,        nucleotides. Thus in another embodiment, the nucleic acid        molecules can have a 3′ overhang of 2 nucleotides, such as TT.        The overhanging nucleotides can be either RNA or DNA.    -   B. Using any method known in the art, compare the potential        targets to the appropriate genome database (human, mouse, rat,        etc.) and eliminate from consideration any target sequences with        significant homology to other coding sequences. One such method        for such sequence homology searches is known as BLAST, which is        available at the National Center for Biotechnology Information        web site of the National Institutes of Health.    -   C. Select one or more sequences that meet your criteria for        evaluation.        Further general information about the design and use of siRNA        can be found in “The siRNA User Guide,” available at the web        site of the laboratory of Dr. Thomas Tuschl at Rockefeller        University.

Negative control siRNAs should have the same nucleotide composition asthe selected siRNA, but without significant sequence complementarity tothe appropriate genome. Such negative controls can be designed byrandomly scrambling the nucleotide sequence of the selected siRNA; ahomology search can be performed to ensure that the negative controllacks homology to any other gene in the appropriate genome. In addition,negative control siRNAs can be designed by introducing one or more basemismatches into the sequence.

The nucleic acid compositions of the invention include both unmodifiedTEF siRNAs and modified TEF siRNAs as known in the art, such ascrosslinked siRNA derivatives as described in U.S. Provisional PatentApplication 60/413,529, which is incorporated herein by reference in itsentirety. Crosslinking can be employed to alter the pharmacokinetics ofthe composition, for example, to increase half-life in the body. Thus,the invention includes siRNA derivatives that include siRNA having twocomplementary strands of nucleic acid, such that the two strands arecrosslinked. For example, a 3′ OH terminus of one of the strands can bemodified, or the two strands can be crosslinked and modified at the 3′OHterminus. The siRNA derivative can contain a single crosslink (e.g., apsoralen crosslink). In some embodiments, the siRNA derivative has atits 3′ terminus a biotin molecule (e.g., a photocleavable biotin), apeptide (e.g., a Tat peptide), a nanoparticle, a peptidomimetic, organiccompounds (e.g., a dye such as a fluorescent dye), or dendrimer.Modifying SiRNA derivatives in this way may improve cellular uptake orenhance cellular targeting activities of the resulting siRNA derivativeas compared to the corresponding siRNA, are useful for tracing the siRNAderivative in the cell, or improve the stability of the siRNA derivativecompared to the corresponding siRNA.

The nucleic acid compositions of the invention can be unconjugated orcan be conjugated to another moiety, such as a nanoparticle, to enhancea property of the compositions, e.g., a pharmacokinetic parameter suchas absorption, efficacy, bioavailability, and/or half-life. Theconjugation can be accomplished by methods known in the art, e.g., usingthe methods of Lambert et al., Drug Deliv. Rev.: 47(1), 99-112 (2001)(describes nucleic acids loaded to polyalkylcyanoacrylate (PACA)nanoparticles); Fattal et al., J. Control Release 53(1-3):137-43 (1998)(describes nucleic acids bound to nanoparticles); Schwab et al., Ann.Oncol. 5 Suppl. 4:55-8 (1994) (describes nucleic acids linked tointercalating agents, hydrophobic groups, polycations or PACAnanoparticles); and Godard et al., Eur. J. Biochem. 232(2):404-10 (1995)(describes nucleic acids linked to nanoparticles).

The nucleic acid molecules of the present invention can also be labeledusing any method known in the art; for instance, the nucleic acidcompositions can be labeled with a fluorophore, e.g., Cy3, fluorescein,or rhodamine. The labeling can be carried out using a kit, e.g., theSILENCER™ siRNA labeling kit (Ambion). Additionally, the siRNA can beradiolabeled, e.g., using ³H, ³²P, or other appropriate isotope. ThedsRNA molecules of the present invention can comprise the followingsequences as one of their strands, and the corresponding sequences ofallelic variants thereof:

hCycT1 ds (SEQ ID NO: 25) 5′-UCCCUUCCUGAUACUAGAAdTdT-3′HcycT1 mm (neg. ctrl) (SEQ ID NO: 26) 5′-UCCCUUCCGUAUACUAGAAdTdT-3′CDK9 ds (SEQ ID NO: 27) 5′-CCAAAGCUUCCCCCUAUAAdTdT-3′CDK9 mm (neg. ctrl) (SEQ ID NO: 28) 5′-CCAAAGCUCUCCCCUAUAAdTdT-3′Spt5 ds (SEQ ID NO: 29) 5′- AACTGGGCGAGTATTACATGAdTdT-3′Spt5 mm (neg. ctrl) (SEQ ID NO: 30) 5′- AACTGGGCGGATATTACATGAdTdT-3′

The above sequences (e.g., sense sequences) correspond to targetedportions of their target mRNAs, as described herein. Reversecomplementary sequences (e.g., antisense sequences) can be generatedaccording to to art recognized principles. dsRNA molecules of thepresent invention preferably comprise one sense sequence or strand andone respective antisense sequence or strand.

Moreover, because RNAi is believed to progress via at least one singlestranded RNA intermediate, the skilled artisan will appreciate thatss-siRNAs (e.g., the antisense strand of a ds-siRNA) can also bedesigned as described herein and utilized according to the claimedmethodologies.

3. Methods of Treatment

The present invention provides for both prophylactic and therapeuticmethods of treating a subject at risk of (or susceptible to) a disorderor having a disorder associated with aberrant or unwanted TEF expressionor activity, e.g., CDK9, CycT1, Spt4, Spt5, or Spt6 activity. As usedherein, the term “treatment” is defined as the application oradministration of the siRNA compositions of the present invention to anindividual, e.g., a patient or subject, or application or administrationof a therapeutic composition including the siRNA compositions to anisolated tissue or cell line from an individual who has a disease, asymptom of a disease, or a predisposition toward a disease, with thepurpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate,improve, or affect the disease, the symptoms of disease, or thepredisposition toward disease. The treatment can include administeringsiRNAs to one or more target sites on one or both of the P-TEFbsubunits, e.g., CDK9 or CycT1, to one or more target sites on one orboth of the DSIF subunits, e.g., Spt5 or Spt4, or to target sites onSpt6, as well as siRNAs to other TEFs. The mixture of different siRNAscan be administered together or sequentially, and the mixture can bevaried over time.

With regards to both prophylactic and therapeutic methods of treatment,such treatments can be specifically tailored or modified, based onknowledge obtained from the field of genomics, particularly genomicstechnologies such as gene sequencing, statistical genetics, and geneexpression analysis, as applied to a patient's genes. Thus, anotheraspect of the invention provides methods for tailoring an individual'sprophylactic or therapeutic treatment with the siRNA compositions of thepresent invention according to that individual's genotype; e.g., bydetermining the exact sequence of the patient's CDK9, CycT1, Spt4, Spt5,and/or Spt6, and designing, using the present methods, an siRNA moleculecustomized for that patient. This allows a clinician or physician totailor prophylactic or therapeutic treatments to patients to enhance theeffectiveness or efficacy of the present methods. Also with regards toboth prophylactic and therapeutic methods of treatment, such treatmentscan be specifically tailored or modified, based on knowledge obtainedfrom the field of pharmacogenomics. In one aspect, the inventionprovides a method for treating a subject having a disease, disorder, orcondition associated with an aberrant or unwanted TEF expression oractivity, e.g. CDK9, CycT1, Spt4, Spt5, or Spt6 expression or activity,by administering to the subject a composition including a CDK9, CycT1,Spt4, Spt5, and/or Spt6 siRNA. Subjects having a disease which is causedor contributed to by aberrant or unwanted CDK9, CycT1, Spt4, Spt5, orSpt6 expression or activity can be identified by, for example, any or acombination of diagnostic or prognostic assays known in the art or asdescribed herein. Administration of a composition including a CDK9,CycT1, Spt4, Spt5, or Spt6 siRNA can occur prior to the manifestation ofsymptoms characteristic of the CDK9, CycT1, Spt4, Spt5, or Spt6aberrance, such that the disease, disorder, or condition is treated orinhibited.

In one aspect, the invention provides a method for preventing in asubject, a disease or condition associated with an aberrant or unwantedCDK9, CycT1, Spt4, Spt5, or Spt6 expression or activity, byadministering to the subject a composition including a CDK9, CycT1,Spt4, Spt5, or Spt6 siRNA. Subjects at risk for a disorder caused orcontributed to by aberrant or unwanted CDK9, CycT1, Spt4, Spt5, or Spt6expression or activity can be identified by, for example, any or acombination of diagnostic or prognostic assays known in the art or asdescribed herein. Administration of a prophylactic agent can occur priorto the manifestation of symptoms characteristic of the CDK9, CycT1,Spt4, Spt5, or Spt6 aberrance, such that a disease or disorder isprevented or, alternatively, delayed in its progression.

Additionally, TEF molecules, e.g. CDK9, CycT1, Spt4, Spt5, and/or Spt6may play an important role in the etiology of certain viral diseases,including, but not limited to, Human Immunodeficiency Virus (HIV),Hepatitis B, Hepatitis C, and Herpes Simplex Virus (HSV). P-TEFb siRNAcompositions can be used to treat viral diseases, and in the treatmentof viral infected tissue or virus-associated tissue fibrosis. Inparticular, as described herein, TEF, e.g. CDK9, CycT1, Spt4, Spt5,and/or Spt6, siRNA compositions can be used to treat HIV infections.Also, TEF modulators can be used in the treatment and/or diagnosis ofvirus-associated carcinoma, including hepatocellular cancer.

4. Treating HIV Infection

In one aspect, the present invention is based on the discovery thatspecific reduction of TEF activity, e.g., CDK9, CycT1, Spt4, Spt5 orSpt6 activity, in human cells is non-lethal and can be used to control,e.g., inhibit, Tat transactivation and HIV replication in host cells.While not wishing to be bound by theory, one model for understandingHIV-1 gene regulation is depicted in FIG. 1A and FIG. 11. Briefly, RNApol II containing nonphosphorylated C-terminal domain (CTD) of thelargest subunit (IIA) assembles on the HIV LTR promoter to form apreinitiation complex. TFIIH binds to nonphosphorylated RNA pol II andplays a critical role in transcription initiation and promoterclearance. TFIIH phosphorylates the CTD of the largest subunit of RNApol II and assists in promoter clearance. The TFIIH complex dissociatesfrom TECs 30 to 50 nucleotides after initiation and is not part of theelongation complexes. P-TEFb, composed of CDK9 and cyclin T1, is acomponent of PICs, however, it may not be an active kinase at thisstage. After promoter clearance, DSIF and NELF associate with thetranscription complex during the early elongation stage. Under standardphysiological conditions and in the case of non-HIV-1 LTR promoters,Spt5 is phosphorylated by CDK9 once DSIF/NELF associate with the earlyelongation complex, and this phosphorylation of Spt5 may sufficientlysupport regular transcription elongation. In the presence of DRB, thekinase activity of CDK9 is inhibited and Spt5 cannot be phosphorylatedby P-TEFb. The unphosphorylated form of Spt5 acts as a negativeregulator and causes inhibition of RNA pol II elongation. In contrast tocellular promoters, transcription from the HIV-1 LTR promoter is notefficient and CDK9 is activated by Tat protein. In the absence of Tat,elongation complexes which originated at the HIV-1 promoter meet DSIFand NELF, CDK9 is unable to efficiently phosphorylate Spt5 and, as aresult, elongation is not processive. After the transcription of afunctional TAR RNA structure, Tat binds to TAR and repositions P-TEFb inthe vicinity of the CTD of RNA pol II and Spt5. Hyperphosphorylation ofthe CTD is carried out by P-TEFb after the formation of Tat-TAR-P-TEFbcomplexes. In addition to CTD phosphorylation, Tat also enhances thephosphorylation of Spt5 mediated by P-TEFb, and the phosphorylated formof Spt5 turns DSIF into a positive regulator of transcription elongation(Ping and Rana, J. Biol. Chem., 276:12951-12958 (2001)). Specificreduction in P-TEFb or DSIF activity can be achieved in a number ofdifferent ways, including RNAi, antisense, ribozymes, or small moleculestargeted to one or both subunits of P-TEFb (e.g., CDK9 or CycT1) or DSIF(e.g., Spt4 or Spt5). Specific reduction in Spt6 activity can beachieved in a number of different ways, including RNAi, antisense,ribozymes, or small molecules targeted to Spt6.

5. Treating Cancer

In another aspect, the present invention is based in part on thediscovery that specific reduction of transcription elongation factoractivity in human cells is non-lethal and can be used to regulate theexpression of genes correlated with diseases or disorders characterizedby unwanted or aberrant cellular proliferation or differentiation, todecrease the growth of cancerous cells, and reduce the metastaticactivity of cancerous cells. Examples of proliferative and/ordifferentiative disorders include cancer, e.g., carcinomas, sarcomas,metastatic disorders or hematopoietic neoplastic disorders, e.g.,leukemias, as well as proliferative skin disorders, e.g., psoriasis orhyperkeratosis. Other myeloproliferative disorders include polycythemiavera, myelofibrosis, chronic myelogenous (myelocytic) leukemia, andprimary thrombocythaemia, as well as acute leukemia, especiallyerythroleukemia, and paroxysmal nocturnal haemoglobinuria. Metastatictumors can arise from a multitude of primary tumor types, including butnot limited to those of prostate, colon, lung, breast and liver origin.Specific reduction in transcription elongation factors such as P-TEFb(CDK9/CycT1), DSIF (Spt4/Spt5) or Spt6, can be achieved in a number ofdifferent ways, including the introduction into a cell of RNAi,antisense, ribozyme, dominant negative mutation or sequences containingsuch mutation, or small molecules targeted to the factor, e.g., one orboth subunits of P-TEFb (CDK9/CycT1), one or both subunits of DSIF(e.g., Spt5 or Spt4) or Spt6.

VII. Methods of Introducing RNAs, Vectors, and Host Cells

Physical methods of introducing nucleic acids include injection of asolution containing the RNA, bombardment by particles covered by theRNA, soaking the cell or organism in a solution of the RNA, orelectroporation of cell membranes in the presence of the RNA. A viralconstruct packaged into a viral particle would accomplish both efficientintroduction of an expression construct into the cell and transcriptionof RNA encoded by the expression construct. Other methods known in theart for introducing nucleic acids to cells may be used, such aslipid-mediated carrier transport, chemical-mediated transport, such ascalcium phosphate, and the like. Thus the RNA may be introduced alongwith components that perform one or more of the following activities:enhance RNA uptake by the cell, inhibit annealing of single strands,stabilize the single strands, or other-wise increase inhibition of thetarget gene.

RNA may be directly introduced into the cell (i.e., intracellularly); orintroduced extracellularly into a cavity, interstitial space, into thecirculation of an organism, introduced orally, or may be introduced bybathing a cell or organism in a solution containing the RNA. Vascular orextravascular circulation, the blood or lymph system, and thecerebrospinal fluid are sites where the RNA may be introduced.

The cell with the target gene may be derived from or contained in anyorganism. The organism may a plant, animal, protozoan, bacterium, virus,or fungus. The plant may be a monocot, dicot or gymnosperm; the animalmay be a vertebrate or invertebrate. Preferred microbes are those usedin agriculture or by industry, and those that are pathogenic for plantsor animals. Fungi include organisms in both the mold and yeastmorphologies. Plants include arabidopsis; field crops (e.g., alfalfa,barley, bean, corn, cotton, flax, pea, rape, nice, rye, safflower,sorghum, soybean, sunflower, tobacco, and wheat); vegetable crops (e.g.,asparagus, beet, broccoli, cabbage, carrot, cauliflower, celery,cucumber, eggplant, lettuce, onion, pepper, potato, pumpkin, radish,spinach, squash, taro, tomato, and zucchini); fruit and nut crops (e.g.,almond, apple, apricot, banana, black-berry, blueberry, cacao, cherry,coconut, cranberry, date, faJoa, filbert, grape, grapefr-uit, guava,kiwi, lemon, lime, mango, melon, nectarine, orange, papaya, passionfruit, peach, peanut, pear, pineapple, pistachio, plum, raspberry,strawberry, tangerine, walnut, and watermelon); and ornamentals (e.g.,alder, ash, aspen, azalea, birch, boxwood, camellia, carnation,chrysanthemum, elm, fir, ivy, jasmine, juniper, oak, palm, poplar, pine,redwood, rhododendron, rose, and rubber). Examples of vertebrate animalsinclude fish, mammal, cattle, goat, pig, sheep, rodent, hamster, mouse,rat, primate, and human; invertebrate animals include nematodes, otherworms, drosophila, and other insects.

The cell having the target gene may be from the germ line or somatic,totipotent or pluripotent, dividing or non-dividing, parenchyma orepithelium, immortalized or transformed, or the like. The cell may be astem cell or a differentiated cell. Cell types that are differentiatedinclude adipocytes, fibroblasts, myocytes, cardiomyocytes, endothelium,neurons, glia, blood cells, megakaryocytes, lymphocytes, macrophages,neutrophils, eosinophils, basophils, mast cells, leukocytes,granulocytes, keratinocytes, chondrocytes, osteoblasts, osteoclasts,hepatocytes, and cells of the endocrine or exocrine glands.

Depending on the particular target gene and the dose of double strandedRNA material delivered, this process may provide partial or completeloss of function for the target gene. A reduction or loss of geneexpression in at least 50%, 60%, 70%, 80%, 90%, 95% or 99% or more oftargeted cells is exemplary. Inhibition of gene expression refers to theabsence (or observable decrease) in the level of protein and/or mRNAproduct from a target gene. Specificity refers to the ability to inhibitthe target gene without manifest effects on other genes of the cell. Theconsequences of inhibition can be confirmed by examination of theoutward properties of the cell or organism (as presented below in theexamples) or by biochemical techniques such as RNA solutionhybridization, nuclease protection, Northern hybridization, reversetranscription, gene expression monitoring with a microarray, antibodybinding, enzyme linked immunosorbent assay (ELISA), Western blotting,radioimmunoassay (RIA), other immunoassays, and fluorescence activatedcell analysis (FACS).

For RNA-mediated inhibition in a cell line or whole organism, geneexpression is conveniently assayed by use of a reporter or drugresistance gene whose protein product is easily assayed. Such reportergenes include acetohydroxyacid synthase (AHAS), alkaline phosphatase(AP), beta galactosidase (LacZ), beta glucoronidase (GUS),chloramphenicol acetyltransferase (CAT), green fluorescent protein(GFP), horseradish peroxidase (HRP), luciferase (Luc), nopaline synthase(NOS), octopine synthase (OCS), and derivatives thereof. Multipleselectable markers are available that confer resistance to ampicillin,bleomycin, chloramphenicol, gentarnycin, hygromycin, kanamycin,lincomycin, methotrexate, phosphinothricin, puromycin, and tetracyclin.Depending on the assay, quantitation of the amount of gene expressionallows one to determine a degree of inhibition which is greater than10%, 33%, 50%, 90%, 95% or 99% as compared to a cell not treatedaccording to the present invention. Lower doses of injected material andlonger times after administration of siRNA may result in inhibition in asmaller fraction of cells (e.g., at least 10%, 20%, 50%, 75%, 90%, or95% of targeted cells). Quantitation of gene expression in a cell mayshow similar amounts of inhibition at the level of accumulation oftarget mRNA or translation of target protein. As an example, theefficiency of inhibition may be determined by assessing the amount ofgene product in the cell; mRNA may be detected with a hybridizationprobe having a nucleotide sequence outside the region used for theinhibitory double-stranded RNA, or translated polypeptide may bedetected with an antibody raised against the polypeptide sequence ofthat region.

The RNA may be introduced in an amount which allows delivery of at leastone copy per cell. Higher doses (e.g., at least 5, 10, 100, 500 or 1000copies per cell) of material may yield more effective inhibition; lowerdoses may also be useful for specific applications.

VIII. Methods of Treatment:

The present invention provides for both prophylactic and therapeuticmethods of treating a subject at risk of (or susceptible to) a disorderor having a disorder associated with aberrant or unwanted target geneexpression or activity. “Treatment”, or “treating” as used herein, isdefined as the application or administration of a therapeutic agent(e.g., a siRNA or vector or transgene encoding same) to a patient, orapplication or administration of a therapeutic agent to an isolatedtissue or cell line from a patient, who has a disease or disorder, asymptom of disease or disorder or a predisposition toward a disease ordisorder, with the purpose to cure, heal, alleviate, relieve, alter,remedy, ameliorate, improve or affect the disease or disorder, thesymptoms of the disease or disorder, or the predisposition towarddisease.

With regards to both prophylactic and therapeutic methods of treatment,such treatments may be specifically tailored or modified, based onknowledge obtained from the field of pharmacogenomics.“Pharmacogenomics”, as used herein, refers to the application ofgenomics technologies such as gene sequencing, statistical genetics, andgene expression analysis to drugs in clinical development and on themarket. More specifically, the term refers the study of how a patient'sgenes determine his or her response to a drug (e.g., a patient's “drugresponse phenotype”, or “drug response genotype”). Thus, another aspectof the invention provides methods for tailoring an individual'sprophylactic or therapeutic treatment with either the target genemolecules of the present invention or target gene modulators accordingto that individual's drug response genotype. Pharmacogenomics allows aclinician or physician to target prophylactic or therapeutic treatmentsto patients who will most benefit from the treatment and to avoidtreatment of patients who will experience toxic drug-related sideeffects.

1. Prophylactic Methods

In one aspect, the invention provides a method for preventing in asubject, a disease or condition associated with an aberrant or unwantedtarget gene expression or activity, by administering to the subject atherapeutic agent (e.g., a siRNA or vector or transgene encoding same).Subjects at risk for a disease which is caused or contributed to byaberrant or unwanted target gene expression or activity can beidentified by, for example, any or a combination of diagnostic orprognostic assays as described herein. Administration of a prophylacticagent can occur prior to the manifestation of symptoms characteristic ofthe target gene aberrancy, such that a disease or disorder is preventedor, alternatively, delayed in its progression. Depending on the type oftarget gene aberrancy, for example, a target gene, target gene agonistor target gene antagonist agent can be used for treating the subject.The appropriate agent can be determined based on screening assaysdescribed herein.

2. Therapeutic Methods

Another aspect of the invention pertains to methods of modulating targetgene expression, protein expression or activity for therapeuticpurposes. Accordingly, in an exemplary embodiment, the modulatory methodof the invention involves contacting a cell capable of expressing targetgene with a therapeutic agent (e.g., a siRNA or vector or transgeneencoding same) that is specific for the target gene or protein (e.g., isspecific for the mRNA encoded by said gene or specifying the amino acidsequence of said protein) such that expression or one or more of theactivities of target protein is modulated. These modulatory methods canbe performed in vitro (e.g., by culturing the cell with the agent) or,alternatively, in vivo (e.g., by administering the agent to a subject).As such, the present invention provides methods of treating anindividual afflicted with a disease or disorder characterized byaberrant or unwanted expression or activity of a target gene polypeptideor nucleic acid molecule. Inhibition of target gene activity isdesirable in situations in which target gene is abnormally unregulatedand/or in which decreased target gene activity is likely to have abeneficial effect.

3. Pharmacogenomics

The therapeutic agents (e.g., a siRNA or vector or transgene encodingsame) of the invention can be administered to individuals to treat(prophylactically or therapeutically) disorders associated with aberrantor unwanted target gene activity. In conjunction with such treatment,pharmacogenomics (i.e., the study of the relationship between anindividual's genotype and that individual's response to a foreigncompound or drug) may be considered. Differences in metabolism oftherapeutics can lead to severe toxicity or therapeutic failure byaltering the relation between dose and blood concentration of thepharmacologically active drug. Thus, a physician or clinician mayconsider applying knowledge obtained in relevant pharmacogenomicsstudies in determining whether to administer a therapeutic agent as wellas tailoring the dosage and/or therapeutic regimen of treatment with atherapeutic agent.

Pharmacogenomics deals with clinically significant hereditary variationsin the response to drugs due to altered drug disposition and abnormalaction in affected persons. See, for example, Eichelbaum, M. et al.(1996) Clin. Exp. Pharmacol. Physiol. 23(10-11): 983-985 and Linder, M.W. et al. (1997) Clin. Chem. 43(2):254-266. In general, two types ofpharmacogenetic conditions can be differentiated. Genetic conditionstransmitted as a single factor altering the way drugs act on the body(altered drug action) or genetic conditions transmitted as singlefactors altering the way the body acts on drugs (altered drugmetabolism). These pharmacogenetic conditions can occur either as raregenetic defects or as naturally-occurring polymorphisms. For example,glucose-6-phosphate dehydrogenase deficiency (G6PD) is a commoninherited enzymopathy in which the main clinical complication ishaemolysis after ingestion of oxidant drugs (anti-malarials,sulfonamides, analgesics, nitrofurans) and consumption of fava beans.

One pharmacogenomics approach to identifying genes that predict drugresponse, known as “a genome-wide association”, relies primarily on ahigh-resolution map of the human genome consisting of already knowngene-related markers (e.g., a “bi-allelic” gene marker map whichconsists of 60,000-100,000 polymorphic or variable sites on the humangenome, each of which has two variants.) Such a high-resolution geneticmap can be compared to a map of the genome of each of a statisticallysignificant number of patients taking part in a Phase II/III drug trialto identify markers associated with a particular observed drug responseor side effect. Alternatively, such a high resolution map can begenerated from a combination of some ten-million known single nucleotidepolymorphisms (SNPs) in the human genome. As used herein, a “SNP” is acommon alteration that occurs in a single nucleotide base in a stretchof DNA. For example, a SNP may occur once per every 1000 bases of DNA. ASNP may be involved in a disease process, however, the vast majority maynot be disease-associated. Given a genetic map based on the occurrenceof such SNPs, individuals can be grouped into genetic categoriesdepending on a particular pattern of SNPs in their individual genome. Insuch a manner, treatment regimens can be tailored to groups ofgenetically similar individuals, taking into account traits that may becommon among such genetically similar individuals.

Alternatively, a method termed the “candidate gene approach”, can beutilized to identify genes that predict drug response. According to thismethod, if a gene that encodes a drugs target is known (e.g., a targetgene polypeptide of the present invention), all common variants of thatgene can be fairly easily identified in the population and it can bedetermined if having one version of the gene versus another isassociated with a particular drug response.

As an illustrative embodiment, the activity of drug metabolizing enzymesis a major determinant of both the intensity and duration of drugaction. The discovery of genetic polymorphisms of drug metabolizingenzymes (e.g., N-acetyltransferase 2 (NAT 2) and cytochrome P450 enzymesCYP2D6 and CYP2C19) has provided an explanation as to why some patientsdo not obtain the expected drug effects or show exaggerated drugresponse and serious toxicity after taking the standard and safe dose ofa drug. These polymorphisms are expressed in two phenotypes in thepopulation, the extensive metabolizer (EM) and poor metabolizer (PM).The prevalence of PM is different among different populations. Forexample, the gene coding for CYP2D6 is highly polymorphic and severalmutations have been identified in PM, which all lead to the absence offunctional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C19 quitefrequently experience exaggerated drug response and side effects whenthey receive standard doses. If a metabolite is the active therapeuticmoiety, PM show no therapeutic response, as demonstrated for theanalgesic effect of codeine mediated by its CYP2D6-formed metabolitemorphine. The other extreme are the so called ultra-rapid metabolizerswho do not respond to standard doses. Recently, the molecular basis ofultra-rapid metabolism has been identified to be due to CYP2D6 geneamplification.

Alternatively, a method termed the “gene expression profiling”, can beutilized to identify genes that predict drug response. For example, thegene expression of an animal dosed with a therapeutic agent of thepresent invention can give an indication whether gene pathways relatedto toxicity have been turned on.

Information generated from more than one of the above pharmacogenomicsapproaches can be used to determine appropriate dosage and treatmentregimens for prophylactic or therapeutic treatment an individual. Thisknowledge, when applied to dosing or drug selection, can avoid adversereactions or therapeutic failure and thus enhance therapeutic orprophylactic efficiency when treating a subject with a therapeuticagent, as described herein.

Therapeutic agents can be tested in an appropriate animal model. Forexample, an siRNA (or expression vector or transgene encoding same) asdescribed herein can be used in an animal model to determine theefficacy, toxicity, or side effects of treatment with said agent.Alternatively, a therapeutic agent can be used in an animal model todetermine the mechanism of action of such an agent. For example, anagent can be used in an animal model to determine the efficacy,toxicity, or side effects of treatment with such an agent.

Alternatively, an agent can be used in an animal model to determine themechanism of action of such an agent.

4. Disease Indications

The compositions of the invention can act as novel therapeutic agentsfor controlling one or more of cellular proliferative and/ordifferentiative disorders, disorders associated with bone metabolism,immune disorders, hematopoietic disorders, cardiovascular disorders,liver disorders, viral diseases, pain or metabolic disorders.

Examples of cellular proliferative and/or differentiative disordersinclude cancer, e.g., carcinoma, sarcoma, metastatic disorders orhematopoietic neoplastic disorders, e.g., leukemias. A metastatic tumorcan arise from a multitude of primary tumor types, including but notlimited to those of prostate, colon, lung, breast and liver origin.

As used herein, the terms “cancer,” “hyperproliferative,” and“neoplastic” refer to cells having the capacity for autonomous growth,i.e., an abnormal state or condition characterized by rapidlyproliferating cell growth. Hyperproliferative and neoplastic diseasestates may be categorized as pathologic, i.e., characterizing orconstituting a disease state, or may be categorized as non-pathologic,i.e., a deviation from normal but not associated with a disease state.The term is meant to include all types of cancerous growths or oncogenicprocesses, metastatic tissues or malignantly transformed cells, tissues,or organs, irrespective of histopathologic type or stage ofinvasiveness. “Pathologic hyperproliferative” cells occur in diseasestates characterized by malignant tumor growth. Examples ofnon-pathologic hyperproliferative cells include proliferation of cellsassociated with wound repair.

The terms “cancer” or “neoplasms” include malignancies of the variousorgan systems, such as affecting lung, breast, thyroid, lymphoid,gastrointestinal, and genito-urinary tract, as well as adenocarcinomaswhich include malignancies such as most colon cancers, renal-cellcarcinoma, prostate cancer and/or testicular tumors, non-small cellcarcinoma of the lung, cancer of the small intestine and cancer of theesophagus.

The term “carcinoma” is art recognized and refers to malignancies ofepithelial or endocrine tissues including respiratory system carcinomas,gastrointestinal system carcinomas, genitourinary system carcinomas,testicular carcinomas, breast carcinomas, prostatic carcinomas,endocrine system carcinomas, and melanomas. Exemplary carcinomas includethose forming from tissue of the cervix, lung, prostate, breast, headand neck, colon and ovary. The term also includes carcinosarcomas, e.g.,which include malignant tumors composed of carcinomatous and sarcomatoustissues. An “adenocarcinoma” refers to a carcinoma derived fromglandular tissue or in which the tumor cells form recognizable glandularstructures.

The term “sarcoma” is art recognized and refers to malignant tumors ofmesenchymal derivation.

Additional examples of proliferative disorders include hematopoieticneoplastic disorders. As used herein, the term “hematopoietic neoplasticdisorders” includes diseases involving hyperplastic/neoplastic cells ofhematopoietic origin, e.g., arising from myeloid, lymphoid or erythroidlineages, or precursor cells thereof. Preferably, the diseases arisefrom poorly differentiated acute leukemias, e.g., erythroblasticleukemia and acute megakaryoblastic leukemia. Additional exemplarymyeloid disorders include, but are not limited to, acute promyeloidleukemia (APML), acute myelogenous leukemia (AML) and chronicmyelogenous leukemia (CML) (reviewed in Vaickus, L. (1991) Crit Rev. inOncol./Hemotol. 11:267-97); lymphoid malignancies include, but are notlimited to acute lymphoblastic leukemia (ALL) which includes B-lineageALL and T-lineage ALL, chronic lymphocytic leukemia (CLL),prolymphocytic leukemia (PLL), hairy cell leukemia (HLL) andWaldenstrom's macroglobulinemia (WM). Additional forms of malignantlymphomas include, but are not limited to non-Hodgkin lymphoma andvariants thereof, peripheral T cell lymphomas, adult T cellleukemia/lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), largegranular lymphocytic leukemia (LGF), Hodgkin's disease andReed-Sternberg disease.

In general, the compositions of the invention are designed to targetgenes associated with particular disorders. Examples of such genesassociated with proliferative disorders that can be targeted includeactivated ras, p53, BRCA-1, and BRCA-2. Other specific genes that can betargeted are those associated with amyotrophic lateral sclerosis (ALS;e.g., superoxide dismutase-1 (SOD1)); Huntington's disease (e.g.,huntingtin), Parkinson's disease (parkin), and genes associated withautosomal dominant disorders.

The compositions of the invention can be used to treat a variety ofimmune disorders, in particular those associated with overexpression ofa gene or expression of a mutant gene. Examples of hematopoieticdisorders or diseases include, but are not limited to, autoimmunediseases (including, for example, diabetes mellitus, arthritis(including rheumatoid arthritis, juvenile rheumatoid arthritis,osteoarthritis, psoriatic arthritis), multiple sclerosis,encephalomyelitis, myasthenia gravis, systemic lupus erythematosis,autoimmune thyroiditis, dermatitis (including atopic dermatitis andeczematous dermatitis), psoriasis, Sjögren's Syndrome, Crohn's disease,aphthous ulcer, iritis, conjunctivitis, keratoconjunctivitis, ulcerativecolitis, asthma, allergic asthma, cutaneous lupus erythematosus,scleroderma, vaginitis, proctitis, drug eruptions, leprosy reversalreactions, erythema nodosum leprosum, autoimmune uveitis, allergicencephalomyelitis, acute necrotizing hemorrhagic encephalopathy,idiopathic bilateral progressive sensorineural hearing loss, aplasticanemia, pure red cell anemia, idiopathic thrombocytopenia,polychondritis, Wegener's granulomatosis, chronic active hepatitis,Stevens-Johnson syndrome, idiopathic sprue, lichen planus, Graves'disease, sarcoidosis, primary biliary cirrhosis, uveitis posterior, andinterstitial lung fibrosis), graft-versus-host disease, cases oftransplantation, and allergy such as, atopic allergy.

Examples of disorders involving the heart or “cardiovascular disorder”include, but are not limited to, a disease, disorder, or state involvingthe cardiovascular system, e.g., the heart, the blood vessels, and/orthe blood. A cardiovascular disorder can be caused by an imbalance inarterial pressure, a malfunction of the heart, or an occlusion of ablood vessel, e.g., by a thrombus. Examples of such disorders includehypertension, atherosclerosis, coronary artery spasm, congestive heartfailure, coronary artery disease, valvular disease, arrhythmias, andcardiomyopathies.

Disorders which may be treated by methods described herein include, butare not limited to, disorders associated with an accumulation in theliver of fibrous tissue, such as that resulting from an imbalancebetween production and degradation of the extracellular matrixaccompanied by the collapse and condensation of preexisting fibers.

Additionally, molecules of the invention can be used to treat viraldiseases, including but not limited to hepatitis B, hepatitis C, herpessimplex virus (HSV), HIV-AIDS, poliovirus, and smallpox virus. Moleculesof the invention are engineered as described herein to target expressedsequences of a virus, thus ameliorating viral activity and replication.The molecules can be used in the treatment and/or diagnosis of viralinfected tissue. Also, such molecules can be used in the treatment ofvirus-associated carcinoma, such as hepatocellular cancer.

IX. Pharmaceutical Compositions

The invention pertains to uses of the above-described agents fortherapeutic treatments as described infra. Accordingly, the modulatorsof the present invention can be incorporated into pharmaceuticalcompositions suitable for administration. Such compositions typicallycomprise the nucleic acid molecule, protein, antibody, or modulatorycompound and a pharmaceutically acceptable carrier. As used herein thelanguage “pharmaceutically acceptable carrier” is intended to includeany and all solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic and absorption delaying agents, and thelike, compatible with pharmaceutical administration. The use of suchmedia and agents for pharmaceutically active substances is well known inthe art. Except insofar as any conventional media or agent isincompatible with the active compound, use thereof in the compositionsis contemplated. Supplementary active compounds can also be incorporatedinto the compositions.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, intraperitoneal, intramuscular, oral (e.g., inhalation),transdermal (topical), and transmucosal administration. Solutions orsuspensions used for parenteral, intradermal, or subcutaneousapplication can include the following components: a sterile diluent suchas water for injection, saline solution, fixed oils, polyethyleneglycols, glycerine, propylene glycol or other synthetic solvents;antibacterial agents such as benzyl alcohol or methyl parabens;antioxidants such as ascorbic acid or sodium bisulfite; chelating agentssuch as ethylenediaminetetraacetic acid; buffers such as acetates,citrates or phosphates and agents for the adjustment of tonicity such assodium chloride or dextrose. pH can be adjusted with acids or bases,such as hydrochloric acid or sodium hydroxide. The parenteralpreparation can be enclosed in ampoules, disposable syringes or multipledose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyetheylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying which yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. They can be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Oral compositions can also be preparedusing a fluid carrier for use as a mouthwash, wherein the compound inthe fluid carrier is applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved, and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Compounds that exhibit large therapeutic indices are preferred. Althoughcompounds that exhibit toxic side effects may be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage touninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the EC50 (i.e., the concentration ofthe test compound which achieves a half-maximal response) as determinedin cell culture. Such information can be used to more accuratelydetermine useful doses in humans. Levels in plasma may be measured, forexample, by high performance liquid chromatography.

A therapeutically effective amount of a composition containing acompound of the invention (e.g., a siRNA, candidate siRNA derivative,modified siRNA, etc.) (i.e., an effective dosage) is an amount thatinhibits expression of the polypeptide encoded by the target gene by atleast 30 percent. Higher percentages of inhibition, e.g., 45, 50, 75,85, 90 percent or higher may be preferred in certain embodiments.Exemplary doses include milligram or microgram amounts of the moleculeper kilogram of subject or sample weight (e.g., about 1 microgram perkilogram to about 500 milligrams per kilogram, about 100 micrograms perkilogram to about 5 milligrams per kilogram, or about 1 microgram perkilogram to about 50 micrograms per kilogram. The compositions can beadministered one time per week for between about 1 to 10 weeks, e.g.,between 2 to 8 weeks, or between about 3 to 7 weeks, or for about 4, 5,or 6 weeks. The skilled artisan will appreciate that certain factors mayinfluence the dosage and timing required to effectively treat a subject,including but not limited to the severity of the disease or disorder,previous treatments, the general health and/or age of the subject, andother diseases present. Moreover, treatment of a subject with atherapeutically effective amount of a composition can include a singletreatment or a series of treatments.

It is furthermore understood that appropriate doses of a compositiondepend upon the potency of composition with respect to the expression oractivity to be modulated. When one or more of these molecules is to beadministered to an animal (e.g., a human) to modulate expression oractivity of a polypeptide or nucleic acid of the invention, a physician,veterinarian, or researcher may, for example, prescribe a relatively lowdose at first, subsequently increasing the dose until an appropriateresponse is obtained. In addition, it is understood that the specificdose level for any particular subject will depend upon a variety offactors including the activity of the specific compound employed, theage, body weight, general health, gender, and diet of the subject, thetime of administration, the route of administration, the rate ofexcretion, any drug combination, and the degree of expression oractivity to be modulated.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

X. Knockout and/or Knockdown Cells or Organisms

A further preferred use for the siRNA molecules of the present invention(or vectors or transgenes encoding same) is a functional analysis to becarried out in eukaryotic cells, or eukaryotic non-human organisms,preferably mammalian cells or organisms and most preferably human cells,e.g. cell lines such as HeLa or 293 or rodents, e.g. rats and mice. Byadministering a suitable siRNA molecules which is sufficientlycomplementary to a target mRNA sequence to direct target-specific RNAinterference, a specific knockout or knockdown phenotype can be obtainedin a target cell, e.g. in cell culture or in a target organism.

Thus, a further subject matter of the invention is a eukaryotic cell ora eukaryotic non-human organism exhibiting a target gene-specificknockout or knockdown phenotype comprising a fully or at least partiallydeficient expression of at least one endogeneous target gene whereinsaid cell or organism is transfected with at least one vector comprisingDNA encoding a siRNA molecule capable of inhibiting the expression ofthe target gene. It should be noted that the present invention allows atarget-specific knockout or knockdown of several different endogeneousgenes due to the specificity of the siRNAi.

Gene-specific knockout or knockdown phenotypes of cells or non-humanorganisms, particularly of human cells or non-human mammals may be usedin analytic to procedures, e.g. in the functional and/or phenotypicalanalysis of complex physiological processes such as analysis of geneexpression profiles and/or proteomes. Preferably the analysis is carriedout by high throughput methods using oligonucleotide based chips.

Using RNAi based knockout or knockdown technologies, the expression ofan endogeneous target gene may be inhibited in a target cell or a targetorganism. The endogeneous gene may be complemented by an exogenoustarget nucleic acid coding for the target protein or a variant ormutated form of the target protein, e.g. a gene or a DNA, which mayoptionally be fused to a further nucleic acid sequence encoding adetectable peptide or polypeptide, e.g. an affinity tag, particularly amultiple affinity tag.

Variants or mutated forms of the target gene differ from the endogeneoustarget gene in that they encode a gene product which differs from theendogeneous gene product on the amino acid level by substitutions,insertions and/or deletions of single or multiple amino acids. Thevariants or mutated forms may have the same biological activity as theendogeneous target gene. On the other hand, the variant or mutatedtarget gene may also have a biological activity, which differs from thebiological activity of the endogeneous target gene, e.g. a partiallydeleted activity, a completely deleted activity, an enhanced activityetc. The complementation may be accomplished by compressing thepolypeptide encoded by the endogeneous nucleic acid, e.g. a fusionprotein comprising the target protein and the affinity tag and thedouble stranded RNA molecule for knocking out the endogeneous gene inthe target cell. This compression may be accomplished by using asuitable expression vector expressing both the polypeptide encoded bythe endogenous nucleic acid, e.g. the tag-modified target protein andthe double stranded RNA molecule or alternatively by using a combinationof expression vectors. Proteins and protein complexes which aresynthesized de novo in the target cell will contain the exogenous geneproduct, e.g., the modified fusion protein. In order to avoidsuppression of the exogenous gene product by the siRNAi molecule, thenucleotide sequence encoding the exogenous nucleic acid may be alteredat the DNA level (with or without causing mutations on the amino acidlevel) in the part of the sequence which so is homologous to the siRNAmolecule. Alternatively, the endogeneous target gene may be complementedby corresponding nucleotide sequences from other species, e.g. frommouse.

XI. Functional Genomics and/or Proteomics

Preferred applications for the cell or organism of the invention is theanalysis of gene expression profiles and/or proteomes. In an especiallypreferred embodiment an analysis of a variant or mutant form of one orseveral target proteins is carried out, wherein said variant or mutantforms are reintroduced into the cell or organism by an exogenous targetnucleic acid as described above. The combination of knockout of anendogeneous gene and rescue by using mutated, e.g. partially deletedexogenous target has advantages compared to the use of a knockout cell.Further, this method is particularly suitable for identifying functionaldomains of the targeted protein. In a further preferred embodiment acomparison, e.g. of gene expression profiles and/or proteomes and/orphenotypic characteristics of at least two cells or organisms is carriedout. These organisms are selected from: (i) a control cell or controlorganism without target gene inhibition, (ii) a cell or organism withtarget gene inhibition and (iii) a cell or organism with target geneinhibition plus target gene complementation by an exogenous targetnucleic acid.

Furthermore, the RNA knockout complementation method may be used for ispreparative purposes, e.g. for the affinity purification of proteins orprotein complexes from eukaryotic cells, particularly mammalian cellsand more particularly human cells. In this embodiment of the invention,the exogenous target nucleic acid preferably codes for a target proteinwhich is fused to art affinity tag. This method is suitable forfunctional proteome analysis in mammalian cells, particularly humancells.

Another utility of the present invention could be a method ofidentifying gene function in an organism comprising the use of siRNA toinhibit the activity of a target gene of previously unknown function.Instead of the time consuming and laborious isolation of mutants bytraditional genetic screening, functional genomics would envisiondetermining the function of uncharacterized genes by employing theinvention to reduce the amount and/or alter the timing of target geneactivity. The invention could be used in determining potential targetsfor pharmaceutics, understanding normal and pathological eventsassociated with development, determining signaling pathways responsiblefor postnatal development/aging, and the like. The increasing speed ofacquiring nucleotide sequence information from genomic and expressedgene sources, including total sequences for the yeast, D. melanogaster,and C. elegans genomes, can be coupled with the invention to determinegene function in an organism (e.g., nematode). The preference ofdifferent organisms to use particular codons, searching sequencedatabases for related gene products, correlating the linkage map ofgenetic traits with the physical map from which the nucleotide sequencesare derived, and artificial intelligence methods may be used to defineputative open reading frames from the nucleotide sequences acquired insuch sequencing projects. A simple assay would be to inhibit geneexpression according to the partial sequence available from an expressedsequence tag (EST). Functional alterations in growth, development,metabolism, disease resistance, or other biological processes would beindicative of the normal role of the EST's gene product.

The ease with which RNA can be introduced into an intact cell/organismcontaining the target gene allows the present invention to be used inhigh throughput screening (HTS). Solutions containing siRNAs that arecapable of inhibiting the different expressed genes can be placed intoindividual wells positioned on a microtiter plate as an ordered array,and intact cells/organisms in each well can be assayed for any changesor modifications in behavior or development due to inhibition of targetgene activity. The amplified RNA can be fed directly to, injected into,the cell/organism containing the target gene. Alternatively, the siRNAcan be produced from a vector, as described herein. Vectors can beinjected into, the cell/organism containing the target gene. Thefunction of the target gene can be assayed from the effects it has onthe cell/organism when gene activity is inhibited. This screening couldbe amenable to small subjects that can be processed in large number, forexample: arabidopsis, bacteria, drosophila, fungi, nematodes, viruses,zebrafish, and tissue culture cells derived from mammals. A nematode orother organism that produces a colorimetric, fluorogenic, or luminescentsignal in response to a regulated promoter (e.g., transfected with areporter gene construct) can be assayed in an HTS format.

The present invention may be useful in allowing the inhibition ofessential genes. Such genes may be required for cell or organismviability at only particular stages of development or cellularcompartments. The functional equivalent of conditional mutations may beproduced by inhibiting activity of the target gene when or where it isnot required for viability. The invention allows addition of siRNA atspecific times of development and locations in the organism withoutintroducing permanent mutations into the target genome.

XII. Screening Assays

The methods of the invention are also suitable for use in methods toidentify and/or characterize potential pharmacological agents, e.g.identifying new pharmacological agents from a collection of testsubstances and/or characterizing mechanisms of action and/or sideeffects of known pharmacological agents.

Thus, the present invention also relates to a system for identifyingand/or characterizing pharmacological agents acting on at least onetarget protein comprising: (a) a eukaryotic cell or a eukaryoticnon-human organism capable of expressing at least one endogeneous targetgene coding for said so target protein, (b) at least one siRNA moleculecapable of inhibiting the expression of said at least one endogeneoustarget gene, and (c) a test substance or a collection of test substanceswherein pharmacological properties of said test substance or saidcollection are to be identified and/or characterized. Further, thesystem as described above preferably comprises: (d) at least oneexogenous target nucleic acid coding for the target protein or a variantor mutated form of the target protein wherein said exogenous targetnucleic acid differs from the endogeneous target gene on the nucleicacid level such that the expression of the exogenous target nucleic acidis substantially less inhibited by the siRNA molecule than theexpression of the endogeneous target gene.

The test compounds of the present invention can be obtained using any ofthe numerous approaches in combinatorial library methods known in theart, including: biological libraries; spatially addressable parallelsolid phase or solution phase libraries; synthetic library methodsrequiring deconvolution; the ‘one-bead one-compound’ library method; andsynthetic library methods using affinity chromatography selection. Thebiological library approach is limited to peptide libraries, while theother four approaches are applicable to peptide, non-peptide oligomer orsmall molecule libraries of compounds (Lam, K. S. (1997) Anticancer DrugDes. 12:145).

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad.Sci. U.S.A. 90:6909; Erb et al. (1994) Proc. Natl. Acad. Sci. USA91:11422; Zuckermann et al. (1994). J. Med. Chem. 37:2678; Cho et al.(1993) Science 261:1303; Carrell et al. (1994) Angew. Chem. Int. Ed.Engl. 33:2059; Carell et al. (1994) Angew. Chem. Int. Ed. Engl. 33:2061;and in Gallop et al. (1994) J. Med. Chem. 37:1233.

Libraries of compounds may be presented in solution (e.g., Houghten(1992) Biotechniques 13:412-421), or on beads (Lam (1991) Nature354:82-84), chips (Fodor (1993) Nature 364:555-556), bacteria (LadnerU.S. Pat. No. 5,223,409), spores (Ladner U.S. Pat. No. '409), plasmids(Cull et al. (1992) Proc Natl Acad Sci USA 89:1865-1869) or on phage(Scott and Smith (1990) Science 249:386-390); (Devlin (1990) Science249:404-406); (Cwirla et al. (1990) Proc. Natl. Acad. Sci.87:6378-6382); (Felici (1991) J. Mol. Biol. 222:301-310); (Ladnersupra.)).

In a preferred embodiment, the library is a natural product library,e.g., a library produced by a bacterial, fungal, or yeast culture. Inanother preferred embodiment, the library is a synthetic compoundlibrary.

XIII. Uses of siRNA Derivatives to Induce RNAi

An siRNA derivative, introduced into cells or whole organisms asdescribed herein, will associate with endogenous protein components ofthe RNAi pathway to bind to and target a specific mRNA sequence forcleavage and destruction. In this fashion, the mRNA to be targeted bythe siRNA derivative will be depleted from the cell or organism, leadingto a decrease in the concentration of the protein encoded by that mRNAin the cell or organism.

For example, one may be seeking to discover a small molecule thatreduces the activity of a kinase whose overexpression leads tounrestrained cell proliferation. This kinase is overexpressed in avariety of cancer cells. A key question to be determined is whether ornot decreasing the activity of this kinase would have unexpecteddeleterious effects on a cell. By expressing an siRNA derivative thattargets for destruction by the RNAi pathway the mRNA encoding the kinasein a cell, the deleterious effects of such a potential drug can bedetermined. That is, the method described here will allow rapidassessment of the suitability of the kinase as a drug target. Oneadvantage of using an siRNA derivative over a conventional siRNA is thatthe siRNA derivative can be more stable, thus the effect of sustainedexposure of a cell to a decrease in expression of a targeted gene can beassessed.

RNAi provides a new approach for elucidation of gene function.RNAi-mediated gene knockdown is useful for genome-wide analysis of genefunction as well as target validation of potentially therapeutic genes.siRNAs are a useful tool for cell biologists studying mammalian genefunction. For example, siRNAs are useful for the analysis of generalcell biological mechanisms such as mitosis, processing and traffic ofRNA transcripts, the formation of cellular junctions, and membranetrafficking. Reagents that can be used for such analyses (e.g. siRNAderivatives that have increased stability in a cell compared to theircorresponding, unmodified siRNA) have commercial value for use in suchresearch.

A selected gene can be knocked down by use of an siRNA and the resultantphenotype can be observed. However, knockdown of an essential gene couldbe lethal or toxic and may affect many pathways in the cell. Therefore,in some cases it is desirable to provide to the cell an siRNA that isnot maximally efficient at knockdown (i.e., inhibiting expression of theprotein translated from the targeted sequence). The adverse effects ofan overly efficient knockdown can be modulated by contacting the cellwith an siRNA derivative that has reduced RNAi activity compared to acorresponding siRNA. Suitable concentrations of an siRNA derivative usedfor this purpose include concentrations that do not maximally inhibitRNAi activity and ameliorate the undesirable effect of the siRNA. Anamount of an siRNA derivative that can cause knockdown with lessefficiency than a corresponding siRNA can be determined using the dualfluorescence assay described herein by incubating an amount of siRNAderivative targeted to a hybrid reporter gene and detecting the amountof inhibition of reporter gene expression. If desired, the level offluorescence can be compared to that in a corresponding dualfluorescence reporter assay in which the corresponding siRNA was usedinstead of the siRNA derivative. In some cases, a useful siRNAderivative is one that inhibits RNAi by less than 100%. For example, ansiRNA derivative that is useful for reducing the RNAi effect of an siRNAcan inhibit RNAi activity by less than, e.g., 90%, 75%, 50%, 25%, or10%.

This invention is further illustrated by the following examples whichshould not be construed as limiting. The contents of all references,patents and published patent applications cited throughout thisapplication are incorporated herein by reference.

EXAMPLES

Examples I-VII demonstrate that the status of the 5′ hydroxyl terminusof the antisense strand determines RNAi activity, whereas a 3′ terminusblock is well tolerated in vivo. Isolation of siRNA from human cellsrevealed that 5′ hydroxyl termini of antisense strands werephosphorylated and 3′ end biotin groups were not efficiently removed.There was no requirement for a perfect A-form helix in siRNA forinterference effects, but an A-form structure was required forantisense-target RNA duplexes. Strikingly, cross-linking of the siRNAduplex by psoralen did not completely block RNA interference, indicatingthat complete unwinding of the siRNA helix is not necessary for RNAiactivity in vivo. These results highlight the importance of 5′ hydroxylin the antisense strand of siRNA, which is essential to initiate theRNAi pathway, and suggest a model where RNA amplification byRNA-dependent RNA polymerase is not essential for RNAi in human cells.

Example I: Dual Fluorescence Reporter System for RNAi Analysis inMammalian Cells

To explore the functional anatomy of siRNA in mammalian cells, a dualfluorescence reporter system was established using HeLa cells as a modelsystem. Two reporter plasmids were used: pEGFP-C1 and pDsRed1-N1,harboring enhanced green fluorescent protein (GFP) or coral (Discosomaspp.)-derived red fluorescent protein (RFP), respectively. Theexpression of these reporter genes was under cytomegalovirus promotercontrol and could be easily visualized by fluorescence microscopy inliving cells. The siRNA sequence targeting GFP was from position 238-258relative to the start codon, and the RFP siRNA sequence was fromposition 277-297 relative to the start codon (FIG. 1A). Usinglipofectamine, HeLa cells were cotransfected with pEGFP-C1 andpDsRed1-N1 expression plasmids and siRNA duplex, targeting either GFP orRFP. Fluorescence imaging was used to monitor GFP and RFP expressionlevels. As shown in FIG. 1B (panels a and b), mock treatment (withoutsiRNA) allowed efficient expression of both GFP and RFP in living cells.Transfection of cells with siRNA duplex targeting GFP (GFP ds)significantly reduced GFP expression (FIG. 1B, panel c), but had noeffect on RFP expression (FIG. 1B, panel d) compared with mock-treatedcells (FIG. 1B, panels a and b). By contrast, transfection of cells withsiRNA duplex targeted to RFP (RFP ds) significantly interfered with theexpression of RFP, but not GFP (FIG. 1B, panels e and f).

To quantify RNAi effects, lysates were prepared from siRNAduplex-treated cells at 42 hours post transfection. GFP and RFPfluorescence in clear lysates was measured on a fluorescencespectrophotometer. The peak at 507 nm (FIG. 1C, left panel) representsthe fluorescence intensity of GFP, and the peak at 583 nm (FIG. 1C,right panel) represents the fluorescence intensity of RFP. GFPfluorescence intensity of GFP ds-treated cells (FIG. 1C, left panel,green line) was only 5% of mock-treated (black line) or RFP ds-treatedcells (cyan line). In contrast to GFP fluorescence, RFP fluorescenceintensity (FIG. 1C, right panel) significantly decreased only in cellstreated with RFP ds (red line), indicating the specificity of the RNAieffect.

To confirm these findings on RNAi effects in living mammalian cells,Western blotting experiments were performed using anti-GFP and anti-RFPantibodies. Analysis of immunoblots revealed that the siRNA targetingGFP inhibited only GFP expression without affecting RFP levels (FIG. 1E,lanes 9-14); siRNA targeting RFP was similarly specific against RFPexpression (FIG. 1F, lanes 9-14). This RNA interference effect dependedon the presence of 21-nucleotide duplex siRNA, but not of the antisensestrand siRNA (FIGS. 1E and F; compare right and left panels). Theseresults demonstrate a reliable and quantitative system for studyingspecific RNA interference in HeLa cells.

Example II: Kinetics of RNA Interference in HeLa Cells

One of the many intriguing features of gene silencing by RNAinterference is its unusually high efficiency—a few dsRNA moleculessuffice to inactivate a continuously transcribed target mRNA for longperiods of time. It has been demonstrated in plants (Cogoni and Macino,1999; Dalmay et al., 2000) and worms (Grishok et al., 2000) that thisinactivation can spread throughout the organism and is often heritableto the next generation. Mutations in genes encoding a protein related toRdRP affect RNAi-type processes in Neurospora (QDE-1; Cogoni and Macino,1999), C. elegans (EGO-1; Smardon et al., 2000) and plants ([SGS2;Mourrain et al., 2000] and [SDE-1; Dalmay et al., 2000]). Theinvolvement of RdRP in amplifying RNAi has been postulated (Lipardi etal., 2001).

To understand the kinetics of gene suppression and persistence of RNAinterference in HeLa cells, lysates were prepared from cellscotransfected with GFP siRNA and dual fluorescence reporter plasmids,pEGFP-C1 and pDsRed1-N1. In this experiment, GFP was the target of theduplex siRNA, while RFP was used as a control for transfectionefficiency and specificity of RNA interference. Emission spectra of GFPin cell lysates at various times after transfection (FIG. 1G,Supplementary Material) show that siRNA duplex caused an RNAinterference effect as early as 6 hours post transfection. This effectgradually increased with time, peaking at 42 hours, then started todecrease at 66 hours (FIG. 1G, green lines). As a control experiment,GFP expression in the presence of antisense strand was also monitoredand showed no RNAi effects (FIG. 1G, blue lines). Thus, RNA interferencecan last for at least 66 hours in HeLa cells (FIG. 1G, green lines).

To quantify the kinetics of RNA interference, the fluorescence intensityratio of target (GFP) to control (RFP) fluorophore in the presence ofsiRNA duplex (ds) was measured and normalized it to the ratio observedin the presence of antisense strand siRNA (as). Normalized ratios lessthan 1.0 indicate specific interference. As shown in FIG. 1D, at 6 hourspost transfection GFP duplex siRNA (green bars) inhibits 60% of GFPexpression compared to antisense strand siRNA (blue bars). RNAinterference reached its maximum (92% inhibition) at 42 hours posttransfection; only 8% of normal GFP expression was observed in duplexsiRNA-treated cells. These results show that RNA interference cansuppress target protein expression up to 66 h, although maximumactivities were observed at 42-54 h post transfection.

Example III: Free 5′ OH Groups on the Antisense Strand of the siRNADuplex are Required for RNA Interference In Vivo

Synthetic 21-nucleotide siRNA duplexes with 5′ hydroxyl termini and 3′overhang have been shown to specifically suppress expression ofendogenous and heterologous genes in Drosophila extracts (Elbashir etal., 2001b) and mammalian cell lines (Elbashir et al., 2001a).Nonetheless, native siRNA, processed by Dicer cleavage of dsRNA,contains 5′ phosphate ends (Elbashir et al., 2001b). It has beendemonstrated in vitro that Drosophila embryo lysates contain a potentkinase activity that phosphorylates the 5′ hydroxyl termini of syntheticsiRNAs (Nykanen et al., 2001). The 5′ phosphate is required on the siRNAstrand that guides target cleavage in RNA interference (Nykanen et al.,2001).

To examine the importance of 5′ termini of siRNA in RNA interference inhuman cells, synthetic siRNAs targeting GFP were modified by using anamino group with a 3-carbon linker (5′ N3, FIG. 2A) to block their 5′termini. Synthetic siRNAs with this modification lacked a hydroxyl groupto be phosphorylated by kinases in vivo. This modification could alsoblock access to siRNA by cellular factors that might require recognizingthe 5′ OH termini. Unmodified siRNA strands were annealed with5′-modified strands, producing siRNA duplexes with 5′ modification atonly the sense strand (5′-N3ss/as), at only the antisense strand(ss/5′-N3as) or at both strands (5′-N3ss/5′-N3as) (FIG. 2B). RNAieffects of these siRNA duplexes were analyzed in the dual fluorescencereporter system as described in FIG. 1. 5′ modification of the sensestrand had no effect on RNAi activity (FIG. 3, compare panels b and c),whereas 5′ modification of the antisense strand completely abolished theRNAi effect (FIG. 3, panels d and e; FIGS. 4A and 4B, upper panels).HeLa cells transfected with antisense strand (as) siRNA as controlshowed no RNAi activity (FIG. 3, panel a). These results demonstratethat the 5′ OH in the antisense strand of the siRNA duplex is animportant determinant of RNAi activity in human cells.

Example IV: Blocking the 3′ End of siRNAs has Little Effect on RNAInterference In Vivo

To determine the effect of 3′ OH groups on RNAi activity, siRNA duplexeswere synthesized containing a 3′ end blocked with 3′ puromycin (3′-Pmn,FIG. 2A) or biotin instead of 3′ OH groups on the overhangdeoxythymidine (FIG. 2B). These 3′ end modifications would block anyprocessing of the siRNA duplex that required a free 3′ hydroxyl group.Three combinations of siRNA duplexes were prepared containing 3′puromycin: 3′ blocked at only the sense strand (ss3′-Pmn/as), at onlythe antisense strand (ss/as3′-Pmn), or at both strands(ss3′-Pmn/as3′-Pmn) (FIG. 2B). A siRNA duplex containing biotin at the3′-end of antisense strand (ss/as3′-Biotin) was also prepared. The RNAiactivities of these siRNA duplexes were analyzed in our dualfluorescence reporter system. Results of these experiments indicate thata 3′ block at either the sense or antisense strand of siRNA duplex hadlittle effect on its RNA interference activity (FIG. 3, panels f-i;FIGS. 4A and 4B, middle panels). Furthermore, biotin pull outexperiments showed that the 3′ end biotin groups on the antisense strandwere not efficiently removed during RNAi activities in HeLa cells (FIG.5, see below). Modifications could be introduced in the 3′ overhangswithout affecting siRNA efficacy, suggesting that RNA interference inmammalian cells does not occur through the recently reportedRdRP-dependent degradative PCR mechanism (Lipardi et al., 2001; Sijen etal., 2001), which requires a free 3′ hydroxyl group.

Example V: A-Form Helix of siRNA is Absolutely Required for EffectiveRNA Interference In Vivo

Synthetic and native siRNAs, generated from ATP-dependent cleavage ofdouble strand RNA, have been proposed to act as “guide RNAs” that targetan associated nuclease complex, the RISC (RNA-induced silencingcomplex), to the corresponding mRNA through strand complementarity(Hammond et al., 2000; Nykanen et al., 2001). How are these siRNAduplexes recognized and incorporated into the RISC protein complex?siRNA duplexes are readily characterized by their A-form helix, whichcan be distinguished from the structures of B-form helix DNA andsingle-stranded RNA in the cell. A single mismatch between a target mRNAand its guide strand siRNA completely prevents target RNA cleavage inDrosophila embryo lysates (Elbashir et al., 2001c). Although themechanism of target recognition has not been experimentallydemonstrated, this finding indicates that recognition requires exactcomplementarity between the guide strand and target mRNA.

These observations raise two fundamental questions regarding RNAieffects in vivo: (1) Is an A-form RNA helix required in the siRNAstructure? (2) Is an A-form helix recognized by proteins after theantisense strand of siRNA duplex is hybridized with the target mRNA? Toaddress these questions, three siRNA duplexes were designed containinginternal bulge structures in the RNA helices (FIG. 2B). The A-form RNAhelix has a deep, narrow major groove and a shallow, wide minor groove.More than one nucleotide bulge has been shown to distort RNA helicalstructures, widening the major groove and enhancing accessibility to itsfunctional groups (Neenhold and Rana, 1995; Weeks and Crothers, 1991;Weeks and Crothers, 1993). 2-nt bulges were chosen to generate distortedA-form helices in siRNAs. Mutant siRNA were synthesized by introducingtwo extra nucleotides into the sense or antisense strand of siRNAduplexes. Combining these mutant siRNA strands with original siRNAsequences produced three siRNA duplexes with an internal bulge at onlythe sense strand (ss-bulge/as), at only the antisense strand(ss/as-bulge), or at both strands (ss-bulge/as-bulge) (FIG. 2B). Thisdesign of bulge-containing siRNAs could dissect the requirement for theA-form helix at two different steps of RNA interference: 1) siRNArecognition by RISC, and 2) RISC targeting of mRNA via the guidingsiRNA. siRNA duplexes with an internal bulge at only the sense strand(ss-bulge/as) caused a structural change in the siRNA duplex (animperfect A-form) without affecting the complementarity between targetmRNA and the antisense strand, which acts as the guiding strand in theRNA interference pathway. RNA interference by these siRNA duplexes wasanalyzed and quantified in the dual fluorescence reporter system asdescribed above.

Surprisingly, the siRNA duplex containing a bulge in its sense strandretained most of its RNA interference activity (FIG. 3, compare panels band j; FIGS. 4A and 4B, lower panels, green line and bars), indicatingthat an A-form siRNA helix is not essential for effective RNAinterference in vivo. However, bulges in the antisense strand or bothstrands of duplex siRNA completely abolished RNA interference ability(FIG. 3, panels k and 1; FIGS. 4A and 4B, lower panels, dark and lightblue line and bars), indicating that effective RNA interference in vivoabsolutely requires A-form helix formation between target mRNA and itsguiding antisense strand.

Example VI: 5′ OH Groups on the Antisense Strand of the siRNA Duplex arePhosphorylated In Vivo

To analyze the phosphorylation status of the 5′ termini of siRNA and toprobe the participation of siRNA 3′ termini in the RNA interferencepathway in vivo, HeLa cells were transfected with 21-nt RNAs containingbiotin at the 3′ terminal of the antisense strand (ss/as3′-Biotin) andisolated the biotinylated siRNA at various times after transfection (seeExperimental Procedures). Briefly, streptavidin magnetic beads were usedto pull out biotinylated siRNAs from transfected cells, washed to removeunbound RNA, and split into two aliquots. One aliquot wasdephosphorylated with shrimp alkaline phosphatase (SAP), and the RNA 5′ends labeled with ³²P by T4 polynucleotide kinase (PNK) reaction. Theother aliquot was subjected to 5′ end radiolabeling with polynucleotidekinase without prior dephosphorylation reaction with SAP. RNA wasresolved on 20% polyacrylamide-7M urea gels and visualized byphosphorimager analysis. Cells without siRNA treatment showed nodetectable signal after biotin pull out assay (FIG. 5, lane 4),indicating the absence of non-specific RNA-bead interactions. Efficient5′-end radiolabeling was observed only when RNA was pretreated withphosphatase (compare lanes 5-9 and 10-14), indicating that the 5′termini of siRNA did not contain free OH groups in vivo. Althoughphosphorylating with SAP and quenching the phosphatase reaction byheating resulted in some RNA degradation, the efficiency of the kinasereaction after SAP treatment is obvious. These results indicate that 5′OH groups are phosphorylated in vivo for RNAi activities.

These experiments have three key findings. First, biotinylated-siRNA canbe isolated from HeLa cells at 6 to 54 hours post transfection (FIG. 5,lanes 5-9). The amount of isolated siRNA decreased in a time-dependentmanner, indicating the degradation of siRNA in vivo. The dualfluorescence assays showed that RNA interference mediated by 3′ endbiotinylated siRNA was as effective as unmodified siRNA (FIG. 3, panelsf and b; FIGS. 4A and 4B, middle panel). RNA interference is seen asearly as 6 hours post siRNA transfection and can be maintained for 42hours post transfection. The ability to isolate biotin-RNA from cellsafter RNA interference had been initiated indicates that biotin was notremoved from the RNA and rules out the possibility of siRNA 3′ OHtermini involvement in the RNA interference pathway in human cells.

Second, in this biotin pull out assay, only siRNA with 5′ OH ends can be³²P-labeled by T4 PNK. As shown in FIG. 5, the siRNA without SAPtreatment was not efficiently labeled by T4 PNK (e.g., compare lane 10to lane 5 and lane 11 to lane 6), indicating that the 5′ termini ofsiRNA did not contain free OH groups in vivo. These 5′ terminal groupscan be removed by alkaline phosphatase treatment for subsequentradiolabeling (FIG. 5, lanes 5-9), indicating that the 5′ termini of thesiRNA had been phosphorylated in vivo.

Third, only the antisense strand is recovered by biotin pull out assays.siRNA duplexes were 5′-end labeled with ³²P by T4 PNK, heat denatured(10 min at 95° C.), and analyzed on a polyacrylamide-7M urea denaturinggel. As shown in FIG. 5 (lane 3), two single-stranded RNA speciescorresponding to the sense and biotinylated-antisense strands wereobserved indicating that the siRNA duplexes were fully denatured underthese conditions. Denatured siRNA duplexes contained≈equal molar amountsof the sense and the antisense strands of RNA (FIG. 5, lane 3). Thecells were transfected with duplex siRNA but the major products of theisolated siRNA (FIG. 5, lanes 5-9) by biotin pull out assay exhibitedelectrophoretic mobilities identical to the antisense strand (lane 3),indicating that only biotinylated anti-sense strands were beingrecovered. These results suggest that RISC melts the duplex siRNA andseparates the antisense from the sense strand during RNA interference invivo.

Example VII: Complete Unwinding of siRNA Duplex is not Necessary for RNAInterference Pathway In Vivo

ATP-dependent unwinding of the siRNA duplex in the RISC has beenproposed to activate the complex to generate RISC*, which is competentto mediate RNAi (Nykanen et al., 2001). Although unwinding of siRNA inDrosophila embryo lysates has been demonstrated in the presence of ATP,the efficiency of unwinding seems low since only 5% of unwound siRNA wasdetected (Nykanen et al., 2001).

To examine whether or not the siRNA duplex in human cells is completelyunwound, RNA interference experiments were performed with siRNA duplexescovalently cross-linked by psoralen photochemistry. Psoralens arebifunctional furocoumarins that intercalate between the base pairs ofdouble-stranded nucleic acids and can photoreact with pyrimidine basesto form monoadducts and cross-links (for review see (Cimino et al.,1985)). The structure of the psoralen derivative,4′-(hydroxymethyl)-4,5′,8-trimethylpsoralen (HMT) used in this study isshown in FIG. 6A. Psoralen cross-linking involves two successivephotochemical reactions that take place at the 3,4 or 4′,5′ double bondsof psoralen (Cimino et al., 1985). Upon long wave UV irradiation(320-400 nm), the intercalated psoralen can photoreact with adjacentpyrimidine bases to form either furan-side or pyrone-side monoadducts,which are linked to only one strand of the helix (Cimino et al., 1985).By absorbing a second photon, the furan-side monoadducts can be driveninto diadducts, which are covalently linked to both strands of the helix(Hearst et al., 1984; Kanne et al., 1982). Psoralen cross-link formationoccurs only when psoralen adds to adjacent and opposite pyrimidine basesin the double helix. The reaction is primarily with uracil in nativeRNAs, but reactions with cytidine have also been reported (Lipson etal., 1988; Thompson and Hearst, 1983; Turner and Noller, 1983). Based onpsoralen photoreactivity, three possible psoralen cross-link sites inthe GFP siRNA duplex are shown in FIG. 6B. Note that there is no chancefor all three sites to be cross-linked in one RNA.

Unlike the noncross-linked ds siRNA, the two strands of the cross-linkedsiRNA duplex couldn't separate from each other under denaturingconditions so that the cross-linked siRNA duplex showedcharacteristically retarded mobility in polyacryalmide gelelectrophoresis (PAGE) containing 7M urea (FIG. 6C). Cross-linkingefficiency depended on the psoralen concentration (FIG. 6C, lanes 2 and3). To further verify the presence of cross-links in the RNA helix andrule out the possibility of only monoadduct formation, the psoralencross-links were irradiated with short wave UV (254 nm), which showedphotoreversal of the cross-linked bonds (FIG. 6C, lane 4). Thecross-linked siRNA duplex (FIG. 6C, lane 3, upper band) was excised fromthe gel and purified. As control, the noncross-linked siRNA that wasirradiated with long wave UV (360 nm) (FIG. 6C, lane 3, lower band) wasalso purified by the same method. The structures of the purifiednoncross-linked and psoralen cross-linked siRNA duplexes were confirmedby PAGE containing 7M urea (FIG. 6C, lanes 5 and 6). Fluorescenceimaging of living cells treated with cross-linked siRNA duplex showedthat the siRNA duplex's inability to separate on PAGE did not completelyabolish its RNA interference activity (FIG. 6D, ds-XL). Quantitativeanalysis of GFP fluorescence intensity indicated that cross-linked siRNAretained 30% of its RNAi activity (FIG. 6E, blue line). These resultsdemonstrate that a complete unwinding of the siRNA duplex is notrequired for gene silencing in vivo (see Discussion).

There is a possibility that the psoralen cross-link of RNA can bephotoreversed during transfection, repaired or removed by some unknownmechanism inside the cells, which might cause the partial RNAinterference effect in vivo observed in FIGS. 6D and 6E. To rule outthis possibility, a psoralen cross-linking experiment was performed withsiRNA duplex containing biotin at the 3′ end of the antisense strand.The cross-linked duplex (ss/as3′-Biotin-XL) was isolated and purified asdescribed above and transfected into HeLa cells by lipofectamine.Biotinylated siRNA was isolated from the cells 30 h post transfection bybiotin pull out assay, SAP treated and ³²P-labeled by T4 PNK asdescribed above. The biotinylated siRNA was still cross-linked (FIG. 7,lane 7) at 30 h post transfection. When UV-irradiated (254 nm), thishigher molecular weight siRNA species was converted into two RNA speciescorresponding to sense and antisense strands (FIG. 7, lane 8),indicating the reversibility of the psoralen cross-link. These resultsshow that cross-linked siRNA duplexes can enter the RNAi pathway.

Summary of Examples I-VII

By using a quantitative dual fluorescence-based system, the kinetics anda number of important parameters involved in the RNAi pathway have beendissected in cultured human cells. The results presented in ExamplesI-VII highlight the role of free 5′ end hydroxyl groups and therequirement of an A-form helical structure between the antisense strandand the target mRNA. It was also found that a complete unwinding of thesiRNA helix is not necessary to cause RNAi effects in vivo.

The time-dependent effect of siRNA may reflect a time lag between targetmRNA degradation and the half-life of the existing protein expressedfrom the target gene. This time dependence may also indicate that thesiRNAs need to be processed or assembled into an active complex withcellular factors for effective RNA interference.

Although RNA interference lasted at least 66 hours in HeLa cells,quantitative analysis indicated that inhibition by siRNAs did notpersist. After reaching maximal activity at 42 hours post transfection,RNA interference started to decrease at 54 hours, with only 70%inhibition activity at 66 hours. It was also found that 5-10% proteinexpressed from the genes targeted by siRNA remained at 42 hours posttransfection, but protein amount showed gradual recovery to normallevels between 66 to 90 hours (3 to 4 days) post transfection (Chiu andRana, unpublished results). The recovery of target gene expression alsoindicates that RNA interference by exogenous siRNA duplex does not existforever in mammalian cells. These findings suggest that the proposedamplification system driven by RdRP and present in plants and nematodesmay not exist or has very little effect on siRNA-mediated gene silencingin mammalian cells.

Recent studies have shown that synthetic siRNAs containing 5′-OH terminican successfully induce RNAi effects in Drosophila embryo lysates(Elbashir et al., 2001c; Nykanen et al., 2001) and cultured mammaliancells (Elbashir et al., 2001a). A model involving a 5′ end kinaseactivity necessary for RNA interference has been proposed (Nykanen etal., 2001). However, there is no evidence that the 5′ end hydroxyl isrequired for in vivo interference activity. The above results show thatreplacing the 5′ OH, a kinase target site, with amino groups inhibitedRNAi activity. Further isolation of siRNA by biotin pull out experimentsrevealed that prior phosphatase activity was required for in vitro 5′end radiolabeling by a polynucleotide kinase. Taken together, theseresults provide strong evidence for the requirement of 5′ end kinaseactivity for RNA interference effects in vivo.

What about a free 3′ end for RNAi effects in vivo? An RNA-directed RNApolymerase (RdRP) chain reaction, primed by siRNA, has recently beenproposed to amplify the interference effects of a small amount oftrigger RNA (reviewed in (Nishikura, 2001)). Lipardi et al. (Lipardi etal., 2001) have shown siRNA-primed RNA synthesis in Drosophila embryolysates and suggested that RNAi in Drosophila involves an RdRP wheresiRNA primes the conversion of target RNA to dsRNA. Further evidence ofRdRP involvement in the RNAi pathway in C. elegans has been provided instudies (Sijen et al., 2001) showing target RNA-templated synthesis ofnew dsRNA. These studies highlight the importance of a 3′ hydroxyl inpriming subsequent RdRP reactions. An RdRP homolog has not yet beenidentified in the human genome, suggesting the presence of a separateenzyme that can carry out primer-dependent replication of an RNAtemplate. The above results demonstrate that blocking the 3′ positiondid not significantly affect RNAi activity of siRNA in human cells.Results of kinetic experiments show that the interference effect lastedonly ˜4 days, indicating the absence of an amplification mechanism inhuman cells. In addition, our biotin pull out experiments show that the3′ end biotin groups on the antisense strand were not efficientlyremoved during RNAi activities in HeLa cells. Based on these studies, amodel is proposed where RNA amplification by RNA-dependent RNApolymerase is not essential for RNA interference in mammalian celllines.

It is interesting to note that there was no requirement for a perfectA-form helix in siRNA for interference effects in HeLa cells, but anA-form structure was required for antisense-target RNA duplexes. Theseresults suggest an RNAi mechanism where RISC formation does not involveperfect RNA helix recognition, but RISC* (the asterisk indicates theactive conformation of the complex) assembly requires an A-form helicalstructure.

The most intriguing results were obtained by cross-linking siRNAs andtesting their interference activities in HeLa cells. Psoralencross-linked siRNA duplexes retained 30% of RNA interference activity.This result can be explained by psoralen photocross-linking chemistry.There are three possible sites in the GFP siRNA duplex where psoralencan cross-link, yet the cross-linking reaction is not efficient enoughto create multiple cross-links in a single given siRNA duplex (Cimino etal., 1985; Thompson and Hearst, 1983). Thus, in the purifiedcross-linked siRNA duplex population, about ⅓ had cross-linking at thesite near the 5′ end of the antisense strand, about ⅓ had cross-linkingin the middle region and the rest had cross-linking near the 3′ end ofthe antisense strand.

It has previously been shown that accessibility to the 5′ termini of theantisense strand is required for efficient RNA interference in vivo. 5′phosphorylation of the antisense strand is also required for RNAinterference in vitro (Nykanen et al., 2001). The cleavage site ontarget mRNA has been shown to be determined by the 5′ end position ofthe target-recognizing siRNA (Elbashir et al., 2001c). Based on thesefindings, it is suggested that unwinding of the siRNA duplex would startfrom the 5′ end of the antisense strand, which sets the ruler for targetmRNA cleavage. If cross-linking occurred near the 5′ end of theantisense strand, it would completely prohibit the unwinding of thesiRNA duplex and block access to the 5′ termini of the antisense strand,which would completely abolish the RNAi effect. If cross-linkingoccurred in the middle of siRNA duplex, near the cleavage site of mRNA,it is suggested that although the siRNA duplex could still undergo someunwinding, this cross-link might interfere with the pairing betweentarget mRNA and the guiding siRNA, thus also blocking the RNAi effect.If cross-linking occurred near the 3′ end of the antisense strand, theduplex RNA could unwind, not completely but sufficient for the antisensestrand to hybridize to the target mRNA. It has previously been shownthat blocking either the 3′ end of the antisense strand or the 5′ end ofthe sense strand has no significant effect on its RNAi activity. Itwould thus be reasonable to believe that a siRNA duplex withcross-linking near the 3′ end of the antisense strand may still becompetent in RNA interference. This hypothesis also explains theremaining 30% RNAi activity in the psoralen-cross-linked siRNA duplex.

These results suggest a possible model for the RNAi pathway in humancells. An RNA-protein complex containing siRNA (RISC) is assembledwithout the requirement for an A-form RNA helix and/or a free 3′-OH. The5′-OH of the siRNA duplex is phophorylated by a kinase. Duringactivation of RISC to RISC*, a 5′43′ helicase unwinds the RNA duplex toallow hybridization between the antisense strand of siRNA and the targetRNA. The requirement of a perfect A-form helix at this stage stronglysuggests that another protein (or protein complex) binds this RNAduplex, either in a structural role and/or assisting in the cleavage ofmRNA. A complete unwinding of the siRNA duplex is not required for thisprocess, nor can this interference activity be amplified via the 3′ end.However, unwinding of the duplex up to the cleavage site may benecessary so that the antisense strand can form an A-form helix with thetarget strand for further protein interactions. These results also argueagainst the involvement of RNA amplification mechanism(s) for RNAinterference in human cells.

In summary, the above results provide new insight into the mechanism ofRNAi in mammalian cells, and guide the design of siRNA structures usefulin probing biological questions and in functional genomic studies.

Example VIII: Improved Dual Fluorescence Assay

pDsRed2-N1 (Catalog #6973-1, BD Biosciences Clontech, Palo Alto, Calif.)encodes DeRed2, a DsRed variant that has been engineered for fastermaturation and lower non-specific aggregation. DsRed2, derived form itprogenitor DeRed1, contains six amino acid substitutions: A105V, I161Tand S197A, which result in the more rapid appearance of red fluorescencein transfected cell lines and R2A, K5E and K9T, which prevent theprotein from aggregation. The extinction coefficient of DsRed2 is 43800(M−1 cm−1) and the quantum yield is 0.55, both are showing significantlyincreasing compared to DsRed1. Intensity of red fluorescence in cellstransfected with pDeRed1 and pDeRed2 is shown in FIG. 8A, siRNAtargeting DsRed1-N1 can also targeting DeRed2-N1 mRNA because thesequence are identical in the targeting region of siRNA.

In an improved dual fluorescence reporter assay, EGFP-C1 encodedenhanced green fluorescence protein (GFP), while DsRed2-N1 encoded redfluorescence protein (RFP2) as described above. Using lipofectamine,HeLa cells were cotransfected with pEGFP-C1 and pDsRed2-N1 expressionplasmids and siRNA duplex, targeting either GFP or RFP. To quantify RNAieffects, lysates were prepared from siRNA duplex-treated cells at 42 hrposttransfection. GFP and RFP fluorescence in clear lysates was measuredon a fluorescence spectrophotometer. The peak at 507 nm (FIG. 8B, leftpanel) represents the fluorescence intensity of GFP, and the peak at 583nm (FIG. 8B, right panel) represents the fluorescence intensity of RFP.GFP fluorescence intensity of GFP ds-treated cells (FIG. 8B, left panel,green line) was only 5% of mock-treated (black line) or RFP ds-treatedcells (blue line). In contrast to GFP fluorescence, RFP fluorescenceintensity (FIG. 8B, right panel) significantly decreased only in cellstreated with RFP ds (red line), indicating the specificity of the RNAieffect.

Thus, by using the DsRed2-N1 plasmid for encoding RFP, a much highersignal-to-noise ration is achieved (i.e., a 10 to 20-fold increase insignal when comparing DsRed1-N1 and DsRed2-N1). Moreover, use of theDsRed2-N1 plasmid results in similar fluorescent intensities for RFP asthose seen for cells transfected with EGFP-C1 (i.e., GFP intensities)making comparison in the dual fluorescence assay more practicable.

Example IX: Quantitative Analysis of RNAi Effects in HeLa CellsTransfected with Modified Single-Stranded (Antisense Strand) siRNAs

pEGFP-C1 (as reporter), pDsRed2-N1 (as control) plasmids and variousamount of antisense strand siRNA (as) were cotransfected into HeLa cellsby lipofectamine. Cells were harvested at 42 h after transfection.Fluorescence Intensity of GFP and RFP in total cell lysates weredetected by exciting at 488 and 568 nm, respectively. The fluorescenceintensity ratio of target (GFP) to control (RFP) fluorophore wasdetermined. The data are set forth in FIG. 9A. Modified siRNAs were asfollows: 2′-O-Methyl-modified as siRNAs (as-2′-Ome, lanes 9-12),2′-Fluoro U and C modified as siRNAs (as-2′FU, 2′FC, lanes 13-16), assiRNAs with phosphorothiolates modification at backbone residues(as-P—S-All, anes 17-20) and as siRNAs with phosphorothiolatesmodification at all backbone residues except the bases 9-12 (as-P-S,lanes 21-24). The intensity ratios of GFP to RFP in various treatmentwere normalized to the ratio observed in the mock treated cells. Anormalized ratio of less than 1.0 indicates a specific RNA interferenceeffect. For comparison, results from unmodified antisense RNA (as, lanes4-7) and duplex siRNA (ds, lane 2-3)-treated cells are included. Thesedata show that single stranded siRNA has much lower efficiency thanduplex siRNA in mediating RNAi.

Single stranded RNA corresponding to the GFP antisense sequence with5′-phosphate group was synthesized and purified according toart-recognized methodologies. The fluorescence intensity ratio of target(GFP) to control (RFP) fluorophore was determined (FIG. 9B) in thepresence of various amount of 5′-phosphorylated as siRNA (5′-P-as, lanes7-12). For comparison, results from unmodified antisense RNA (as, 400nM, lane 6) and duplex siRNA (ds, lane 2-5)-treated cells are included.These data show that phosphorylation of single-stranded siRNA (antisensestrand) does not much improve its RNA interference activity.

Example X: Quantitative Analysis of RNAi Effects in HeLa CellsTransfected with Modified Duplex siRNAs

Results set forth in Example II showed that RNAi effects typicallypeaked between 42-54 h post transfection and targeted gene expressionstarted to be restored by 66 h post transfection. To determine if theduration of RNAi could be prolonged by increasing the half life ofsiRNAs, various chemical modifications were made to nucleotides thataffected siRNA stability. These modified siRNAs were then tested in animproved dual fluorescence reporter assay which was set forth in ExampleVIII. The sequence of EGFP siRNA and EGFP mRNA, the specific mRNAcleavage site, plus the structures of the chemically modifiednucleotides are diagrammed in FIG. 1. The specific chemicalmodifications, the particular siRNA strand(s) where modifications weremade, and the effect of the chemically modified siRNA on RNAi activityare summarized in Table 1. RNAi activity of siRNAs was evaluated witheight different siRNA concentrations (ranging from 1-200 nM). Eachexperiment was completed in duplicate and repeated twice.

pEGFP-C1 (as reporter), pDsRed2-N1 (as control) plasmids and variousamount of modified siRNA were cotransfected into HeLa cells bylipofectamine. Cells were harvested at 42 h after transfection.Fluorescence intensity of GFP and RFP in total cell lysates weredetected by exciting at 488 and 568 nm, respectively. The fluorescenceintensity ratio of target (GFP) to control (RFP) fluorophore wasdetermined in the presence of modified siRNAs and normalized to theratio observed in the mock treated cells. A normalized ratio of lessthan 1.0 indicates a specific RNA interference effect. Data arepresented in FIG. 10. For comparison, results from unmodified duplexsiRNA (ds, lane 2-5)-treated cells are included in each panel. Unlessotherwise indicated, all residues are modified.

FIG. 10A depicts the results from cells treated with duplex siRNA with2′-Deoxy modification at internal residues within the sense strand(ss-2′Deoxy/as, lanes 6-11).

An interesting result was seen by modifying the 2′OH to a bulky methylgroup to create 2′OMe nucleotides that were incorporated into sense,antisense or both strands of EGFP siRNAs (FIG. 19). This modificationwas hypothesized to improve RNAi efficacy because 2′OMe groups arethought to increase RNA stability by inducing an altered RNAconformation that is more resistant to nucleases (Cummins et al., 1995).This modification is also thought to increase RNA affinity for RNAtargets and improve hybridization kinetics (Majlessi et al., 1998). FIG.10B depicts results from cells treated with duplex siRNA with2′-O-Methyl modification at internal residues within the sense strand(ss-2′Ome/as, lanes 6-11) or the antisense strand (ss/as-2′-Ome, lanes12-17). Despite the potential benefits, 2′OMe nucleotides incorporatedinto either the sense or antisense strand greatly diminished EGFP genesilencing to ˜25% or ˜16%, respectively, while double-stranded 2′OMemodified siRNAs completely abolished RNAi (FIG. 10B and Table 1, rows12-14). These results suggested that the methyl group, as a bulky group,may severely limit the interactions between siRNAs, target mRNAs and theRNAi machinery required for successfully mediating RNAi. It is worthnoting that since the bulkiness of the methyl group would likely be thecause of decreased RNAi activity rather than the actual lack of the 2′OHspecifically, these studies still supported the conclusion that the 2′OHwas not required for RNAi. The effects of modifying the 2′OH ofnucleotides on RNAi were next studied by replacing uridine and cytidinein the antisense strand of siRNA with 2′-Fluoro-uridine (2′-FU) and2′-Fluoro-cytidine (2′-FC), which have a fluoro-group at the 2′ positionin place of the 2′OH (FIG. 19). Addition of a 2′ fluoro-group shouldincrease the stability of the siRNA by making the siRNAs lessrecognizable to RNases thereby providing siRNAs protection fromdegradation. When measured in the dual fluorescence assay, 2′FU, FCsiRNAs, modified only in the sense strand (ss-2′FU, 2′-FC/as, FIG. 10Clanes 6-15), only in the antisense strand (ss/as-2′-FU,2′-FC, FIG. 10Clanes 16-25), or in both strands (ds-2′FU,2′FC, FIG. 10C lanes 26-35),all showed decreased EGFP fluorescence when normalized to non-targetedRFP fluorescence that was comparable to the normalized decrease seenwith wild type siRNAs (FIG. 10C; Table 1, rows 1-4). These resultssuggested that the 2′OH was not required for RNAi and that nucleotidesmodified with 2′ fluoro-groups could be used in siRNA constructs tosuccessfully induce RNAi-mediated gene silencing.

In a final analysis of modifications that may potentially increase siRNAstability without disrupting RNAi potency, a thioate linkage (P-S) wasintegrated into the backbone of the EGFP siRNA strand(s). P—S linkageswere previously used in antisense methodology for increasing resistanceto ribonucleases (reviewed in (Stein, 1996)) and therefore, werepostulated to enhance the stability of siRNAs. FIG. 10D depicts resultsfrom cells treated with duplex siRNA with phosphorothiolate modificationat each backbone residue of the sense strand (ss-P-S-all/as, lanes6-12), antisense strand (ss/as-P-S-all, lanes 13-22) and both strands(ds-P-S-all, lanes 23-31). FIG. 10E depicts results from cells treatedwith duplex siRNA with phosphorothiolate modification at each backboneresidue of both strands except for bases 9-12 of the antisense strand(ds-P-S, except center region, lanes 15-23). For comparison, cellstreated with duplex siRNA with phosphorothiolate modification at eachbackbone residue of both strand (ds-P-S-all) are also shown (lanes6-14). Incorporating the P-S linkages into the double-stranded siRNAsense strand led to moderate levels of RNAi activity (62% inhibition),while P-S linkages in either the antisense or both strands of the siRNAsled to just less than ˜50% RNAi-induced inhibition (Table 1, rows15-17). These results suggested that the P-S modifications did notprohibit RNAi-mediated degradation and only moderately affected theefficiency of RNAi. Interestingly, incorporating 2′FU, FC modificationsinto the antisense strand in addition to the added P-S linkages showedlower levels of EGFP gene silencing (Table 1, row 18), indicating thatthere was a synergistic effect that decreased but did not inhibitRNAi-mediated degradation when both the 2′ F groups and the P-S linkageswere incorporated into siRNAs.

In summary, these data indicate that 2′ Deoxy modifications within thesense strand are well tolerated, whereas 2′-O-Methyl modification is notwell tolerated (either within the sense or antisense strand). Moreover,2′-FU and 2′-FC modifications are well tolerated within either strand orwithin both strands. Note that siRNA duplexes having every internal Uand C modified with 2′F are virtually as efficient at mediating RNAi asare their unmodified counterparts. Also well tolerated arephosphorothioate linkages between backbone residues of the sense and/orantisense strands. Leaving the most internal residues unmodified induplex siRNA having phosphorothioate linkages between backbone residuesof the sense and antisense strands did not significantly improve theRNAi activity.

Example XI: Kinetics of RNAi Effects of Duplex siRNA with 2′-FluoroUridine and Cytidine Modification in HeLa Cells Showing Effect ofModified siRNA is Much More Persistent than the Unmodified siRNA

To address whether increased stability seen with modified siRNAsprolonged the duration of RNAi in vivo, RNAi, induced by unmodified and2′FU, FC modified double-stranded EGFP siRNAs, was assayed in the dualfluorescence reporter assay over a period of 120 h (FIG. 11). Thefluorescence intensity ratio of target (GFP) to control (RFP) proteinwas determined in the presence of unmodified double-strand (ds) RNA(blue bars) and duplex siRNA with 2′-Fluoro uridine and cytidinemodification (ds-2′FU, 2′FC, cyan bar) and normalized to the ratioobserved in the presence of Mock treated cells (red bars). A normalizedratio of less than 1.0 indicates specific RNA interference.

Although 2′FU, FC modified EGFP siRNAs were slower to show RNAi effectsby 6-18 h, maximal RNAi effects occurred by 42 h post-transfection forboth modified and unmodified siRNAs. The maximal activity for bothsiRNAs was also in the same range, with both showing ˜85-90% inhibitionof GFP expression. However, the RNAi effects observed over the period of66-120 h revealed that the effect of modified siRNAs was much morepersistent than unmodified siRNA. By 120 h post-transfection, the effectof modified siRNAs still remained at ˜80% inhibition of GFP expressionwhile the effect of unmodified siRNAs had dropped to less than ˜40%inhibition. These results strongly indicated that there was a directlink between the duration of the RNAi effects and siRNA stability inhuman cells. Furthermore, these results showed conclusively that siRNAsstabilized by chemical modifications, like the 2′ FU, FC-modifications,can be used to effectively induce and significantly prolongRNAi-mediated gene silencing in vivo.

Example XII: Study of Duplex siRNA Stability in HeLa Cell Lysate

As the data set forth in Example X showed that siRNAs modified withstabilizing 2′-FU, FC groups could effectively mediate RNAi to levelscomparable to wild type, it was necessary to show that thesemodifications did in fact enhance siRNA stability. To measure thestability of siRNA in cell extracts, unmodified or modified EGFPantisense strand siRNA were 5′-labeled with [gamma-³²P] ATP by T4polynucleotide kinases. Duplex siRNAs were formed by annealing an equalmolar ratio of unmodified or modifed sense strand siRNA with the 5′-³²Plabeled antisense strand. 50 μmole duplex siRNA which labeled at 5′ endof the antisense strand were incubated with 500 ug HeLa cytoplasmicextract in 50 ul reaction mixture containing 20 mM Hepes, pH 7.9, 100 mMKCl, 10 mM NaCl, 2 mM MgCl₂, 10% glycerol. After incubation for varioustimes with cell extract, siRNAs were analyzed on 20% polyacrylamide gelcontaining 7M Urea followed by phosphorimage analysis (Fugi). Data arepresented in FIG. 12. FIG. 12A depicts a stability comparison ofunmodified and modified antisense strand siRNA. Unmodifiedsingle-stranded siRNA has a very short half-life in cell extract, thatis 50% of them degraded in <10 min. 2′Fluoro modified single stranddoesn't increase its half life. 2′-Ome modification moderately increasesthe stability of single-stranded siRNA while phosphorothioatemodification within the backbone maintains greater stability of thesingle-stranded siRNA in extracts. FIG. 12B depicts a stabilitycomparison of duplex siRNAs with unmodified and modified antisensestrand. Both 2′-Fluoro and 2′-Ome modification at the antisense strandof the duplex siRNA make the duplex RNA much more stable than theunmodified one. However, phosphorothioates modification at antisensestrand of the duplex seems only have moderate effect. This may be due toan increased RNAse H sensitivity of hybrids formed from unmodified sensestrand and phosphorothioate modified antisense strand. FIG. 12C depictsa stability comparison of duplex siRNAs containing modification at bothstrands. Modification dramatically increase the stability of the duplexsiRNA when made at both strands of the siRNA duplex.

Results from experiments demonstrating similar results are depicted inFIGS. 12D and 12E. FIG. 12D shows the stability of the various 2′FU, FCmodified siRNAs as compared to wild type siRNAs over time. Wild typedouble-stranded siRNAs showed a steady loss of intact siRNAs over thecourse of the experiment, with only ˜7% of the original concentration ofintact siRNAs remaining after 1 h in extract (FIG. 12D; dark blue line).Intact modified or unmodified single stranded antisense siRNAs werequickly lost over the time course and were virtually undetectable by 30min in extract (FIG. 12D; black and red lines). In contrast,double-stranded siRNAs with 2′FU, FC modifications in either theantisense strand or both strands remained predominantly intact over thecourse of the experiment with ˜68 or ˜81%, respectively, of the originalsiRNA population remaining intact throughout the duration of theexperiment (FIG. 12D; green and light blue lines). These resultsindicated that the 2′FU, FC modifications did indeed increase thestability of the siRNAs upon exposure to extract and that having thesemodifications in both strands provided the siRNAs with the moststability.

In a similar experiment, the stability of P-S modified EGFP siRNAs wasevaluated. Unmodified, doubled-stranded antisense siRNAs showed aboutthe same rate of siRNA loss as described in the above experiment (FIG.12E; dark blue lines). However, P-S modified single-stranded antisensesiRNAs showed a markedly increased rate of stability over the course ofthe experiment, showing ˜63% of the original siRNAs remaining intactafter 1 h in extract as compared to 0% intact for single-strandedunmodified antisense siRNAs (FIG. 12E; black and red lines). Stabilityof double-stranded siRNAs with P-S modifications in both strands wascomparable to the stability seen with the modified single-strandedantisense strand with ˜63% of the originally siRNA population remainingintact after 1 h (FIG. 12E; light blue lines). Double-stranded siRNAswith P-S modifications in only the antisense strand showed weaker butstill significant stability with ˜42% of the original siRNA populationremaining intact through to 1 h in extract (FIG. 12E; green lines).These results showed that the P-S modifications increased the stabilityof the siRNAs and most notably, increased the stability of both singleand double stranded siRNAs.

Example XIII: Quantitative Analysis of RNAi Effects of Duplex siRNAswith 2′-Fluoro Uridine and Cytidine Modifications, and 2′-Fluoro Uridineand Cytidine Modifications in Combination with 2′-Deoxy Modifications,in HeLa Cells

Results set forth in Example X indicated that the 2′OH was not requiredfor RNAi and that nucleotides modified with 2′ fluoro-groups could beused in siRNA constructs to successfully induce RNAi-mediated genesilencing. To support the conclusion that the 2′OH was not required forRNAi, adenine and guanine deoxynucleotides that inherently have 2′H inplace of the 2′OH (FIG. 19) were incorporated into the sense, antisense,or both strands of 2′FU FC-modified EGFP siRNAs to determine theireffect on RNAi. This example demonstrates that 2′-OH is not required forsiRNA to enter the RNAi pathway, but that an A-form helix is requiredfor mRNA targeting by siRNA.

pEGFP-C1 (as reporter), pDsRed2-N1 (as control) plasmids and variousamount of modified siRNA were cotransfected into HeLa cells bylipofectamine. Cells were harvested at 42 h after transfection.Fluorescence intensity of GFP and RFP in total cell lysates weredetected by exciting at 488 and 568 nm, respectively. The fluorescenceintensity ratio of target (GFP) to control (RFP) fluorophore wasdetermined in the presence of modified siRNAs and normalized to theratio observed in the mock treated cells.

Modified siRNA duplexes with modifications in the antisense strand atthe 2′ position of the sugar unit are set forth in FIG. 13A andconsisted of the following: 2′-hydroxyl wild type (DS), 2′-deoxymodified as siRNAs (SS/AS-Deoxy), 2′-Fluoro U and C modified as siRNAs(SS/AS-2′FU,FC), 2′-Fluoro U and C and 2′-deoxy A and G at positions 9,10, and 13 modified as siRNAs (SS/AS-2′FU,FC+(9,10,13) dA, dG),2′-Fluoro U and C and 2′-deoxy A and G at positions 9-19 modified assiRNAs (SS/AS-2′FU,FC+(9-19) dA, dG), 2′-Fluoro U and C and 2′-deoxy Aand G at positions 1-13 modified as siRNAs (SS/AS-2′FU,FC+(1-13) dA,dG), and 2′-Fluoro U and C and 2′-deoxy A and G modified as siRNAs(SS/AS-2′FU,FC, dA, dG). The hypothetical cleavage site on the targetmRNA is also depicted. The data from cells treated with duplex siRNAwith modified antisense strands are set forth in FIG. 13B. A normalizedratio of less than 1.0 indicates a specific RNA interference effect. Forcomparison, results from unmodified duplex siRNA (ds, lanes 2-6)-treatedcells are included.

These data indicate that siRNA with 2′-Fluoro modifications at uridineand cytidine (SS/AS-2′ FU,FC, lanes 16-24) is as effective as unmodifiedduplex siRNA in RNA interference, indicating that 2′-OH is not requiredfor siRNA to enter the RNAi pathway. However, 2′-deoxy substitution inthe antisense strand completely bocked siRNA function (SS/AS-2′ deoxy,lanes 7-15). In general, mixing 2′-Fluoro modification with deoxymodification could rescue siRNA function (FIG. 13B, lanes 25-60). When2′FU, FC nucleotides were incorporated into the EGFP siRNA anti-strandwith guanine and adenine deoxynucleotides at positions 9, 10, and 13,which base pair with nucleotides lining the cleavage site, (FIG. 13A),EGFP RNAi effects were almost indistinguishable from wild type levels(FIG. 13B, lanes 25-33; Table 1, row 5). In addition, siRNAs that hadthe entire antisense strand replaced with 2′ FU, 2′ FC, dATP, and dGTPnucleotides still showed moderate levels of RNAi activity at ˜42%, or˜44% if the sense strand was also modified with 2′FU, FC (FIG. 13B,lanes 52-60; Table 1, rows 7, 8).

FIG. 13C depicts siRNA duplexes with modifications in both strands atthe 2′ position of the sugar unit, and consisted of the following:2′-hydroxyl wild type (DS, lanes 2-6), 2′-deoxy modified as siRNAs(SS/AS-Deoxy, lanes 7-15), 2′-Fluoro U and C modified in both strands(SS-2′FU,FC/AS-2′FU,FC, lanes 16-24), 2′-Fluoro U and C modified in bothstrands and 2′-deoxy A and G at positions 9, 10, and 13 within theantisense strand (SS-2′FU,FC/AS-2′FU,FC+(9,10,13) dA, dG, lanes 25-33),2′-Fluoro U and C modified in both strands and 2′-deoxy A and G atpositions 9-19 within the antisense strand (SS-2′FU,FC/AS-2′FU,FC+(9-19)dA, dG, lanes 34-42), 2′-Fluoro U and C modified in both strands and2′-deoxy A and G at positions 1-13 within the antisense strand(SS-2′FU,FC/AS-2′FU,FC+(1-13) dA, dG, lanes 43-51), and 2′-Fluoro U andC modified in both strands and 2′-deoxy A and G within the antisensestrand (SS-2′FU,FC/AS-2′FU,FC, dA, dG, lanes 52-60). Results from cellstreated with duplex siRNA with modifications in both strands as setforth in FIG. 13C are depicted in FIG. 13D and table 1, rows 6, 8, 30,32.

All together, these results demonstrated that a 2′OH group was notrequired for RNAi-mediated degradation and, even more specifically, wasnot required for nucleotides base paired with nucleotides lining themRNA cleavage site. There was, however, a limit on the extent to whichdeoxynucleotides could substitute for ribonucleotides since replacingthe entire siRNA sense strand with deoxynucleotides decreased EGFP genesilencing to ˜38% inhibition and replacing either the antisense strandor both strands entirely with deoxynucleotides completely abolished EGFPRNAi (see FIG. 10, FIG. 13 and Table 1, rows 9-11). Nonetheless, theseresults collectively showed that nucleotides with either 2′F- or 2′Hgroups can selectively replace ribonucleotides within the siRNA sequenceto effectively induce RNAi. These data also further demonstrated that Aform helix formed by pairing between the antisense strand of siRNA andits target mRNA is required for the RISC protein complex to recognizeits target. Furthermore, the data further demonstrated that the 2′OH isnot required for the RISC complex to cleave its target mRNA.

Example XIV: Quantitative Analysis of RNAi Effects of Duplex siRNAs withN3-Methyl Uridine Modifications in HeLa Cells

Data set forth in Example V indicated that the A form helix is requiredfor the mechanism of RNAi, as 2 nt bulges that distort A-form helicesbetween antisense siRNAs and target mRNAs abolished RNAi. To testwhether the major groove of the A form helix was required for RNAi,siRNAs were modified with N³-Methyl Uridine (3 MU) nucleotides thatremove an H-bond donor at N³—H. The structure of N³-Methyl-Uridine (3mU) is depicted in FIG. 14A. Structurally, the bulky N³-Methyl groupwould jut into the major groove of the A-form helix, potentiallyintroducing sterical clash between base pairs. In addition, the presenceof 3 MU in the major groove may also introduce a steric clash betweenRNA and RNA-interacting proteins (Saenger, 1984). Therefore, both sterichindrance and the loss of an H-bond donor by the addition of theN³-Methyl group should destabilize RNA-protein interactions in the majorgroove.

pEGFP-C1 (as reporter), pDsRed2-N1 (as control) plasmids and variousamount of modified siRNA were cotransfected into HeLa cells bylipofectamine. Cells were harvested at 42 h after transfection.Fluorescence intensity of GFP and RFP in total cell lysates weredetected by exciting at 488 and 568 nm, respectively. The fluorescenceintensity ratio of target (GFP) to control (RFP) fluorophore wasdetermined in the presence of modified siRNAs and normalized to theratio observed in the mock treated cells. FIG. 14C depicts the resultsfrom cells treated with duplex siRNA having 3 mU modifications withinthe entire antisense strand (SS/AS-3 mU, lanes 7-15), 3 mU modificationswithin the entire antisense strand and 2′-Fluoro modifications aturidine and cytidine bases within the sense strand (SS-2′FU, FC/AS-3 mU,lanes 16-24), and 3 mU modification at position 11 within the antisensestrand (SS/AS-(11)-3 mU, lanes 25-33). The modified siRNA duplexes wereprepared by annealing modified antisense strand containing single ormultiple 3 mU modifications with unmodified sense strand (SS/AS-(11)-3mU and SS/AS-3 mU) or sense strand having 2′-Fluoro modifications(SS-2′FU, FC/AS-3 mU). For comparison, results from cells treated withunmodified duplex siRNA (ds, lane 2-6) are also shown. 3 MU modifiedEGFP siRNAs introduced into Hela cells completely abolished RNAi (FIG.14C, Table 1, rows 25). RNAi was also abolished if only one 3 MUmodification was introduced specifically at U11 of the antisense strand,which is one of the nucleotides that base pairs with A248 of the targetEGFP mRNA cleavage site (FIGS. 14B and 14C, Table 1, row 26). Theseresults indicated that disrupting the functional groups of the majorgroove of the A-form helix formed by the antisense strand and its targetmRNA specifically at the cleavage site inhibited RNAi. These data alsosuggested that the major groove was required for mediating RNAi and forRNA-RISC* interactions that subsequently lead to mRNA cleavage.

Example XV: Structural Integrity of the 5′ End of the Antisense Strandin siRNA-mRNA Duplexes is More Important for Mediating RNAi than the 3′End

Data set forth in Example VII using psoralen photochemistry suggestedthat complete unwinding of the siRNA duplex is not required for RNAi invivo because psoralen cross-linked siRNAs did not completely abolishgene silencing. These results suggested that a single cross-linkingevent occurring near the 3′ end of the antisense strand still allowedfor the initial unwinding of duplex siRNAs from the 5′ end, freeingenough of the nucleotides in the antisense strand to hybridize to thetarget mRNA and induce RNAi, even if unwinding was not complete. Thelocation of this crosslinking site is indicated by a bar in FIG. 15A. Ifthis were the case, then unwinding of siRNAs must start from the 5′ endof the antisense strand, a conclusion supported by the fact thatblocking either the 3′ end of the antisense siRNA strand or the 5′ endof the sense siRNA strand had no significant effect on RNAi activity(see Examples III and IV). If this 5′ to 3′ unwinding model was correct,sequences near the 3′ end of the antisense siRNA strand or 5′ end of thesense siRNA strand should be changeable without significantlyinterfering with RNAi.

This Example directly tests the model set forth above and demonstratesan aysmmetric requirement for duplex siRNA structure in RNA interferencein vivo. To test this hypothesis, EGFP siRNAs with mismatched base pairsat either the 5′ (nt 1, 2) or 3′ (nt 18, 19) ends were introduced intothe antisense strand (FIG. 15B). pEGFP-C1 (as reporter), pDsRed2-N1 (ascontrol) plasmids and various amount of modified siRNA werecotransfected into HeLa cells by lipofectamine. Cells were harvested at42 h after transfection. Fluorescence intensity of GFP and RFP in totalcell lysates were detected by exciting at 488 and 568 nm, respectively.The fluorescence intensity ratio of target (GFP) to control (RFP)fluorophore was determined in the presence of modified siRNAs andnormalized to the ratio observed in the mock treated cells. FIG. 15Cdepicts results from cells treated with duplex siRNA having mismatcheslocated at the 3′ end [SS/AS-(18,19) mm, lanes 7-14] or 5′ end[SS/AS-(1,2) mm, lanes 15-22] of the antisense strand. For comparison,results from unmodified duplex siRNA-treated cells are also shown (ds,lane 2-6). siRNAs with mismatches near the 5′ end of the antisensestrand showed only ˜35% inhibition in the dual fluorescence reporterassay whereas mismatches at the 3′ end retained a significant level ofgene silencing at ˜77% (FIG. 15C; Table 1, rows 27-28). These resultsstrongly indicated that the integrity at the 5′ end of the antisensestrand in the duplex was functionally more important than the 3′ end.

Further demonstrating this point are data set forth above in ExampleXIII, wherein 2′FU, FC plus dATPs, dGTPs were incorporated into theantisense strand siRNAs predominantly at the 5′ end (nts 1-13) orpredominantly at the 3′ end (nts 9-19) (see FIG. 13C). In the dualfluorescence reporter assay, predominantly 5′ modified antisense[AS-2′FU, FC+(1-13) dA, dG] EGFP siRNAs were only moderately effective,inducing RNAi at ˜43%, or at 45% if the sense strand was also modifiedto 2′FU, FC (see FIG. 13C, Table 1, rows 29-30). However, predominantly3′ modified and 5′ unmodified antisense [AS-2′FU, FC+(9-19) dA, dG]siRNAs significantly induced RNAi activity at ˜91%, or at 64% if thesense strand was also modified to 2′FU, FC (see FIG. 13C, Table 1, rows31-32). These contrasting results suggested that the 5′ region of theantisense strand was more sensitive to modification than the 3′ end. Alltogether, these data suggested that recognition of siRNA duplexes by anas yet unidentified RNA helicase occurs asymmetrically with thestructure of the antisense 5′ end of the duplex preferentiallydistinguished from the 3′ end during the initiation of unwinding.

Example XVI: Modified siRNAs that Stabilize A-U Base Pair Interactionscan Induce RNAi

In addition to incorporating modifications that affected the stabilityof siRNAs, nucleotides chemically modified to strengthen the base pairinteractions between two complementary bases were analyzed. In theory,increasing the stability of base pair interactions may increase thetargeting efficiency of siRNAs to target mRNA sequences.

Increasing targeting efficiency may then induce more robust RNAi effectswith siRNAs that are weaker at binding to their target sequence or havemismatched sequences, and thus, are not showing a high degree of RNAi.

To bolster base pairing interactions, 5-Bromo-uridine (U[5Br]),5-Indo-uridine (U[51]) or 2,6-Diaminopurine (DAP) (FIG. 19), which aremodified nucleotides known to increase the association constant betweenA-U base pairs (Saenger, 1984), were incorporated into siRNAs and testedin the dual fluorescence report assay. Double-stranded siRNAs havingU[5Br], U[5I] or DAP modifications incorporated into the antisensestrand were capable of inducing RNAi activity at levels of ˜70% forU[5Br], ˜59% for U[5I] and ˜51% for DAP (FIG. 16, Table 1, rows 19-21).

Interestingly, when 2′FU, FC stabilizing modifications in the sensestrand were combined with these modifications in the antisense strand,gene silencing was not as efficient as wild type in inducing RNAi. EGFPgene silencing was 31% for the 2′FU, FC-modified sense siRNA base pairedwith U[5Br]-, ˜42% for U[5I]-, or ˜35% for DAP-modified antisense siRNAs(Table 1, rows 22-24). These results suggested that enhancing theinteractions between base pairs through these siRNA modifications was aviable option for increasing mRNA targeting efficiency, but that therewas a limit to how stable the base pairing interactions can be madebefore they interfere with siRNA unwinding.

Example XVII: Modified siRNAs Enter into the RNAi Pathway in HeLa CellLysates

Although the dual fluorescence reporter assay did detect changes in EGFPgene expression with the modified siRNAs created herein, it was possiblethat gene silencing was being induced by a mechanism other thanRNAi-mediated degradative pathways. This Example demonstrates thatmodified siRNA enter into the RNA interference pathway by using an invitro RNAi assay. To test whether the targeted mRNA was indeed beingcleaved upon exposure to modified siRNAs, an in vitro RNAi assay wasperformed to measure the cleavage of a ³²P-cap labeled mRNA target uponincubation with modified siRNAs and HeLa cytoplasmic extract. This invitro RNAi assay is well known in the art. Cleavage products wereresolved on an 8% polyacrylamide-7 M urea gel.

In this assay, 10 nM cap-labeled target RNA was incubated with 100 nMsiRNA having the following modifications within the antisense strand:2′-Fluoro U and C (SS/AS-2′FU,FC), 2′-Fluoro U and C and 2′-deoxy A andG at positions 9, 10 and 13 (SS/AS-2′FU,FC+(9,10,13) dA,dG)), 2′-FluoroU and C and 2′-deoxy at each A and G (SS/AS-2′FU,FC+dA,dG), 2′-deoxy ateach position (SS/AS-2′-deoxy), 2′-OMe at each residue (SS/AS-2′-OMe),P-S at each residue (SS/AS—P-S), 5-Bromo-uridine at each U(SS/AS-U[5Br]), (5-Indo-uridine at each U (SS/AS-U[5I]), DAP at eachpurine (SS/AS-DAP), 3 MU at each U (SS/AS-3 MU), 3 MU at position11(SS/AS-91103 MU), mismatches at position 1 and 2 (SS/AS-(1,2) mm),mismatches at position 18 and 19 (SS/AS-(18,19) mm), 2′-Fluoro U and Cand 2′-deoxy A and G at positions 1-13 (SS/AS-2′FU,FC+(1-13) dA,dG), and2′-Fluoro U and C and 2′-deoxy A and G at positions 9-19(SS/AS-2′FU,FC+(9-19) dA,dG). Reaction products were resolved on an 8%polyacrylamide-7M urea gel.

Results from the assay are depicted in FIG. 17. The arrows indicate thecapped target RNA and the 5′ cleavage product; the resulting 3′ fragmentis unlabeled and is therefore invisible. Mock treated mRNAs did not showan observable cleavage product (FIG. 17, lane 1), but wild type and allmodified siRNAs that displayed gene silencing effects in vivo showedclearly visible cleavage products in vitro (FIG. 17; lanes 2,8-11,14-17). Furthermore, modified siRNAs that did not show any markedgene silencing effects in vivo did not show any distinct cleavageproducts in the in vitro assay (FIG. 17; lanes 1, 6-7, 12-13),suggesting that the cleavage events observed were specifically dependenton functional siRNAs. These in vitro results provided a strongcorrelation between the in vivo gene silencing observed with themodified siRNAs and target mRNA degradation, indicating that themodified siRNAs were distinctly targeting mRNAs for cleavage andsubsequent degradation through the in vivo RNAi pathway.

Summary of Examples VIII-XVII

By introducing various chemical modifications into siRNAs and measuringtheir effects on RNAi, the above examples reveal new insights into themechanism of RNAi and teach new approaches for increasing the efficacyof RNAi in vivo, e.g. in human cells.

The step-wise process of RNAi is depicted in FIG. 18. In the first stepof RNAi induction, the 5′ ends of the siRNA duplex are phosphorylated,resulting in the formation of a siRNA-RISC complex. The data presentedhere showing the asymmetric nature of unwinding then suggests anATP-dependent event during which siRNA is unwound from the 5′ end of theantisense strand and RISC is activated. Following RISC activation, theantisense strand of the unwound siRNA guides the siRNA-RISC* complex tothe target mRNA. The guide antisense strand base pairs with the targetmRNA, forming an A-form helix and the RISC* protein complex recognizesthe major groove of the A-form helix, an event that occurs independentlyof the RNA 2′OH of the guide antisense siRNA. In the final step of thisprocess, the target mRNA is cleaved by RISC*, which is another eventthat occurs independently of the 2′OH of the guide antisense siRNA.RISC* is then recycled to catalyze another cleavage event.

A. The Requirement for the A-Form Helix Supercedes the Requirement forthe 2′OH in RNAi

Several important mechanistic findings were presented here that not onlymore clearly defined the mechanism of the RNAi pathway, but will alsoincrease the utility of RNAi in various applications. That the 2′OH wasnot required for RNAi was the most important of these results as thisdiscovery has several important implications for the structural andcatalytic elements required for the RNAi pathway. Remarkable functionalimplications were that the RNAi machinery does not require the 2′OH forrecognition of siRNAs and the catalytic ribonuclease activity of RISCdoes not involve 2′OH groups of the guide antisense RNA. Anotherconsequence of this discovery was that a variety of chemical groups,including fluoro- or deoxy-groups, could substitute for the 2′OH insiRNAs, indicating that no distinguishing chemical specificity wasrequired for RNAi at the 2′ position. These findings would suggest thatother properties of the siRNA-mRNA duplexes, such as core structuralelements, were essential for siRNA. If helical structure was the key toRNAi induction, then the A-form helix that forms between siRNAs and thetarget mRNA would indeed be required for RNAi, as was previously shown(Chiu and Rana, 2002). Furthermore, the 2′ fluoro- or combined 2′fluoro-, deoxy modified antisense siRNAs lacking the 2′OH would have tocompetently form an A-form helix to induce RNAi as shown here. This willlikely turn out to be the case since 2′ fluoro-modified RNA-RNA hybridswere previously reported to exhibit an A-form helical conformation(Cummins et al., 1995; Luy and Marino, 2001), lending significant meritto the idea that helical structure strongly influences RNAi efficiency.Still another implication of these particular results was that alternatechemical groups at the 2′ position that allow the A-form helix to beretained but help siRNAs evade recognition by RNases can increase siRNAstability and prolong RNAi effects induced in vivo.

It was previously shown in C. elegans and Drosophila extracts thatcompletely substituting one or both siRNA strands with deoxynucleotidesabolished RNAi (Elbashir et al., 2001; Parrish et al., 2000), and thoseobservations were consistent with the data presented here. The failureof true DNA-RNA hybrids to induce RNAi most plausibly relates to theargument that structure, and thus the A-form helix, was an essentialdeterminant for RNAi induction. Based on circular dichroism spectra,DNA-RNA hybrids displayed characteristics that were intermediate betweenA- and B-form helices (Cummins et al., 1995). Following the contentionthat the A-form helix was an absolute requirement for RNAi induction, 2′deoxy siRNA-mRNA target duplexes would not be recognized by the RNAimachinery because they would not be forming the proper A-form helicalstructure. Therefore, RNAi would not be induced by DNA-RNA hybrids, ashas been observed. It is also worth mentioning that microRNAs (miRNAs)induce post-transcriptional gene silencing (PTGS) through the samepathway as RNAi but ultimately, only inhibit translation machineryinstead of inducing RNA degradation, the event that defines RNAi. Theonly observable difference between the two mechanisms is that RNAirequires the A-form helix but miRNA-induced PTGS does not, as miRNAsoften mismatch with their target mRNAs, forming a bulge that woulddistort the helical structure. This would suggest that the differencesbetween the miRNA-induced silencing mechanism and siRNA-mediated RNAimay solely be attributable to differences in RNA-RNA helical structure,and further supported a model in which helical structure was the soledeterminant for whether RNAi was induced.

It was also previously reported that replacement of uridine with 2′ FU,corresponding to ¼ of the bases of long dsRNAs elicited RNAi effects inC. elegans, while deoxycytodine incorporated into long dsRNAs diminishedRNAi effects (Parrish et al., 2000). However, exactly where thesemodified nucleotides fell within the sequence structure of RNAi-inducingsiRNAs and whether these modified nucleotides in the longer RNAscorresponded to the mRNA cleavage site or major groove after beingprocessed to siRNAs was not clear. It has also been reported that siRNAsin which 3′ overhangs and two of the 3′ end ribonucleotides werereplaced with deoxyribonucleotides retained RNAi activity upon exposureto Drosophila extracts (Elbashir et al., 2001). Presumably, replacingtwo of the 3′ end base-paired nucleotides with deoxynucleotides wouldnot disrupt the overall A-form structure of the siRNA-mRNA duplexrequired for RNAi and would thereby allow RNAi induction.

Neither analyses in C. elegans or in Drosophila extracts ascertainedwhether there was a distinct requirement for the 2′ OH for cleavage siterecognition and the cleavage event itself during RNAi induction. Theresults presented here demonstrated that exclusively using 2′FU, FCmodifications in siRNAs and selectively substituting indeoxyribonucleotides for nucleotides base paired with the nucleotideslining the mRNA cleavage site, or even replacing the entire sequence ofsiRNA with a combination of 2′ fluoro- and 2′ deoxy-nucleotides,elicited RNAi induction. Therefore, it has now been definitivelyestablished that recognition of the mRNA-target cleavage site andsubsequent cleavage did not require the 2′OH of the antisense siRNA toinduce RNAi. As a final point, the inhibitory RNAi effects seen with thebulky 2′OMe modification, which was also shown previously withDrosophila (Elbashir et al., 2001), did demonstrate that there weresteric constraints on the types of 2′ modifications that would beamenable for inducing RNAi. As 2′OMe modifications probably did notdisrupt the A-form helix of the siRNA-mRNA duplex (Cummins et al.,1995), the methyl group may be sterically interfering with protein-RNAinteractions thereby preventing RNAi. Nevertheless, steric constraintsnotwithstanding, this analysis conclusively showed that thenon-essential nature of the 2′ position could very much be exploited forimproving the efficacy of RNAi in a variety of applications.

B. Improving the Efficacy of RNAi Using Chemical Modifications

The chemical modifications analyzed improved upon the status quoshort-lived RNAi effects seen in vivo in human cells, significantlyincreasing the duration of RNAi effects typically observed.Modifications like the 2′ fluoro- and P—S linkages both increased thehalf-life of siRNAs upon exposure to cytoplasmic extracts, and in vivostudies with 2′ FU, FC siRNAs showed that increasing the half life ofsiRNAs did in fact prolong the effects of RNAi. This indicated thatshort-lived RNAi effects usually observed in human cells were due atleast in part to the degradation of siRNAs. That the stabilizing siRNAmodifications still allowed for a substantial level of RNAi inductionshowed that these modifications will be invaluable for studying thephenotypic effects of prolonged gene-silencing in cell culture or inincreasing the long-term in vivo effects of siRNAs in clinicalapplications. Interestingly, the P-S-modified, single-stranded antisensestrand did not show increased RNAi effects in the dual fluorescencereporter assay used here (data not shown) despite showing significantlyincreased stability (FIG. 3A (a)). This suggested that stability was notthe main reason why single-stranded antisense RNAi was not as effectivein inducing RNAi as dsRNA. Nonetheless, creating P-S modifications inthe siRNA backbone showed that stabilizing the siRNA backbone did notinhibit RNAi and signified that using chemical modifications thatstabilized phosphate linkages was a viable option for prolonging RNAieffects.

Another option for increasing the efficacy of RNAi was uncovered by theanalysis of modifications that should enhance base pairing interactionsbetween antisense siRNA and targeted mRNA. DAP is a naturally occurringnucleobase that sometimes replaces adenine in phages like the cyanophageS-2L (Kirnos et al., 1977). Incorporation of DAP into RNA strandspromotes the formation of three Watson and Crick hydrogen bonds betweenDAP and uridine, increasing the stability of interactions seen betweenA-U base pairs (Luytena and Herdewijna, 1998). U[5Br] and U[5I] havealso been shown to have higher association constants when base paired toA residues than unmodified uridine (Saenger, 1984). When any of thesemodifications were incorporated into siRNAs, RNAi was still quiteefficient, indicating that modifications that stabilize base pairinginteractions can be used in designing siRNAs for various applications.It was also notable that siRNAs with 2′ Fluoro-modifications introducedinto sense strands and base paired with the DAP, U[Br] or U[5I]antisense strands had decreased RNAi efficiency. 2′ Fluoro-modificationshave been shown to significantly increase the melting temperaturebetween base pairs (Cummins et al., 1995). Consequently, the stabilizingeffect on base pairing interactions when both the 2′ Fluoro- and DAP,U[Br] or U[5I] modifications were present may have actually hindered theunwinding of the siRNA duplex. If the unwinding of the siRNA washindered, then there would be less single antisense siRNAs available toinduce RNAi, accounting for the observed decrease in RNAi activity.

C. Other Structural Determinants for RNAi Induction

Another structural facet of the RNAi mechanism was uncovered using the 3MU modification which showed that the major groove of the A-form helixwas required for RNAi. This finding builds on previous data showing thatthe A-form helix was required for RNAi (Chiu and Rana, 2002). Together,these results suggested that the specific structure of the A-formhelical RNA that forms the major groove and contains the mRNA cleavagesite was important for recognition by the RNAi machinery. Conceivably,RNA-RISC* contacts depend on the structural integrity of the majorgroove for precise interactions and ultimately, to initiate cleavage ofthe target. By disrupting the major groove, RISC* may no longer be ableto interact or only weakly interacts with the siRNA-mRNA target duplexthereby preventing mRNA cleavage. Alternatively, RISC* might still beable to interact with the destabilized RNA helix but not recognize thecleavage site within the major groove as the catalytic site if theconformation of the RNA helix and more specifically the major groove wasaltered.

The other structural property of siRNAs defined by these analyses wasthe asymmetric nature of siRNA unwinding. Initiation of siRNA unwindingfrom the 5′ end was previously suggested from the ability of singlecross-linked siRNAs to still induce RNAi (Chiu and Rana, 2002). Buildingon those studies by stacking mismatched or modified nucleotides oneither the 3′ or 5′ end of the antisense strand to gauge the tolerancefor mismatches or modifications on one end over the other, it was shownhere that RNAi depended on the integrity of the 5′, and not the 3′, endof the antisense strand of the siRNA duplex. These results suggestedthat like RISC*, the RNA helicase, which has not yet been identified,also recognizes structural properties of the siRNA duplex as opposed tospecific sequences of the RNA strands. This recognition appears to beasymmetric with the structure of the antisense 5′ end favored over the3′ end, and is similar to how restriction enzymes can preferentiallycleave the DNA backbone asymmetrically within a palindromic sequence.Further structural analysis of siRNAs to pinpoint what properties of theantisense 5′ end contribute to the asymmetric nature of the duplexshould help elucidate the specific structural elements required forduplex recognition by the RNA helicase for siRNA unwinding. That themodified siRNAs displayed effective RNAi in vivo and in vitro was alsosignificant as it confirmed that the observed gene silencing wasmediated by the RNAi pathway. These results also indicated that usingchemical modifications that allow for efficient RNAi induction shouldwork in the design of any given siRNA to increase its stability andcapacity to specifically induce RNAi in vivo.

Example XVIII: Peptide Modification of 3′ Termini of siRNA

Peptides can be linked to the 3′ terminus of an siRNA. For example, ansiRNA containing NH₂ groups at their 3′ termini can be synthesized usingmethods known in the art and as described herein, thus producing, e.g.,exocyclic amine on protected nucleotides. In an example of a peptidemodification of a 3′ terminus of an siRNA, a Tat-derived peptide (fromamino acids 47-57) was synthesized on solid support (rink amide resin)using standard FastMoc protocols. A cysteine residue was added to theamino terminus of the peptide for conjugation to the RNA. All Fmoc-aminoacids, piperidine, 4-dimethylaminopyridine, dichloromethane, N,N-dimethylforamide, 1-hydroxybenzotriazole (HOBT),2-(1H-benzotriazo-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate(HBTU), diisopropylethylamine, and HMP-linked polystyrene resin wereobtained from Applied Biosystems Division, Perkin Elmer. Trifluoroaceticacid, 1,2-ethanedithiol, phenol, thioanisol were from Sigma. Cleavageand deprotection of the peptide was carried out in 2 ml of Reagent K for6 hours at room temperature. Reagent K contained 1.75 ml TFA, 100 μLthioanisole, 1000 water, and 50 μl of ethanedithiol. After cleavage fromthe resin, peptide was purified by HPLC on a Zorbax 300 SB-C8 column.The mass of fully deprotected and purified peptides was confirmed by FABmass spectrometry.

siRNA containing 3′-end amino groups were synthesized.NHS-ester-maleimide crosslinkers (Pierce) were used for conjugation toTat peptide (amino acids 48-57) and the conjugation reaction was carriedout according to the manufacturer's instructions. The NHS ester moietyof the crosslinker was reacted with the RNA as described herein. Afterpurification on a C18 column, the RNA-NHS-maleimide conjugate was addedto the peptide that contains Cys (0.1 M phosphate, pH 8, roomtemperature, 1 hour). Peptide-RNA conjugate was purified on 7 M-ureadenaturing gels.

Similar methods can be used to attached other compounds, e.g.,nanoparticle-RNA conjugates can be prepared using such methods.

Transfection of the siRNA-peptides was carried out withoutLipofectamine™ or any other transfection reagents. Robust RNAi activitywas observed.

These data demonstrate that modification of the 3′ terminus of siRNAdoes not eliminate the ability of the siRNA derivative to be effectivefor inhibiting expression of a targeted sequence. Furthermore, suchsiRNA derivatives can be used directly for transfection without the useof transfection reagents.

Example XIX: Photocleavable Biotin Modification of 3′ Termini of siRNA

A novel photocleavable biotin was synthesized and attached to the 3′terminus of an siRNA. Briefly, NHS esters of biotin (5 nmole) wereconjugated to free amino groups at the 3′-end of an siRNA duplex (1nmole) in an aqueous solution (e.g., 0.1 M phosphate buffer pH 8 at roomtemp for 1 hour). 3′-end amino RNA was purchased from a commercialsource (Dharmacon). RNA-biotin siRNA was incubated with cell extractsand the RNA-protein complex was isolated using avidin magnetic beads.After adding the mutant competitive non-biotin RNA and followed byextensive washing, RNA-protein complexes were released by long wave UV(360 nm) treatment at room temperature. In previous methods, avidinbeads are heated with SDS to release proteins that also contain a largenumber of bead-binding proteins. The present method allows the isolationof specific siRNA-bound proteins. The structure of the novelphotocleavable biotin is shown in FIG. 20.

Experimental Procedures for Examples I-XIX

siRNA Preparation 21-nucleotide RNAs were chemically synthesized as 2′bis(acetoxyethoxy)-methyl ether-protected oligos by Dharmacon(Lafayette, Colo.). Synthetic oligonucleotides were deprotected,annealed and purified as described by the manufacturer. Successfulduplex formation was confirmed by 20% non-denaturing polyacrylamide gelelectrophoresis (PAGE). All siRNAs were stored in DEPC (0.1% diethylpyrocarbonate)-treated water at −80° C. The sequences of GFP or RFPtarget-specific siRNA duplexes were designed according to themanufacturer's recommendation and subjected to a BLAST search againstthe human genome sequence to ensure that no endogenous genes of thegenome were targeted.Culture and Transfection of Cells

Hela cells were maintained at 37° C. in Dulbecco's modified Eagle'smedium (DMEM, Invitrogen) supplemented with 10% fetal bovine serum(FBS), 100 units/ml penicillin and 100 μg/ml streptomycin (Invitrogen).Cells were regularly passaged at sub-confluence and plated 16 hr beforetransfection at 70% confluency. Lipofectamine (Invitrogen)-mediatedtransient cotransfections of reporter plasmids and siRNAs were performedin duplicate 6-well plates as described by the manufacturer for adherentcell lines. A transfection mixture containing 0.16-0.66 μg pEGFP-C1 and0.33-1.33 μg pDsRed1-N1 reporter plasmids (Clontech), various amounts ofsiRNA (1.0 nM-200 nM), and 10 μl lipofectamine in 1 ml serum-reducedOPTI-MEM (Invitrogen) was added to each well. Cells were incubated intransfection mixture for 6 hours and further cultured in antibiotic-freeDMEM. Cells were treated under same conditions without siRNA for mockexperiments. At various time intervals, the transfected cells werewashed twice with phosphate buffered saline (PBS, Invitrogen), flashfrozen in liquid nitrogen, and stored at −80° C. for reporter geneassays.

In Vivo Fluorescence Analysis

pEGFP-C1, pDsRed1-N1 reporter plasmids and 50 nM siRNA werecotransfected into HeLa cells by lipofectamine as described above exceptthat cells were cultured on 35 mm plates with glass bottoms (MatTekCorporation, Ashland Mass.) instead of standard 6-well plates.Fluorescence in living cells was visualized 48 hours post transfectionby conventional fluorescence microscopy (Zeiss). For GFP and RFPfluorescence detection, FITC and CY3 filters were used, respectively.

Dual Fluorescence Reporter Gene Assays

pEGFP-C1, pDsRed1-N1 reporter plasmids and 50 nM siRNA werecotransfected into HeLa cells. EGFP-C1 encoded enhanced greenfluorescence protein (GFP), while DsRed1-N1 encoded red fluorescenceprotein (RFP). Cells were harvested as described above and lysed inice-cold reporter lysis buffer (Promega) containing protease inhibitor(complete, EDTA-free, 1 tablet/10 ml buffer, Roche MolecularBiochemicals). After clearing the resulting lysates by centrifugation,protein in the clear lysate was quantified by Dc protein assay kit(Bio-Rad). 120 μg of total cell lysate in 160 μl reporter lysis bufferwas measured by fluorescence spectrophometry (Photo TechnologyInternational). The slit widths were set at 4 nm for both excitation andemission. All experiments were carried out at room temperature.Fluorescence of GFP in cell lysates was detected by exciting at 488 nmand recording from 498-650 nm. The spectrum peak at 507 nm representsthe fluorescence intensity of GFP. Fluorescence of RFP in the same celllysates was detected by exciting at 568 nm and recording from 588 nm-650nm; the spectrum peak at 583 nm represents the fluorescence intensity ofRFP. The fluorescence intensity ratio of target (GFP) to control (RFP)fluorophore was determined in the presence of siRNA duplex andnormalized to that observed in the presence of antisense strand siRNA.Normalized ratios less than 1.0 indicate specific interference.

Improved Dual Fluorescence Assay

HeLa cells were maintained at 37° C. in Dulbecco's modified Eagle'smedium (DMEM, Invitrogen) supplemented with 10% fetal bovine serum(FBS), 100 units/ml penicillin, and 100 μg/ml streptomycin (Invitrogen).Cells were regularly passaged at subconfluence and plated 16 hr beforetransfection at 70% confluency. Lipofectamine (Invitrogen)-mediatedtransient cotransfections of reporter plasmids and siRNAs were performedin duplicate 6-well plates. A transfection mixture containing 0.16 μgpEGFP-C1 and 0.33 μg pDsRed2-N1 reporter plasmids (Clontech), variousamount of siRNA (From 0.5 nM to 400 nM), and 10 μl lipofectamine in 1 mlserum-reduced OPTI-MEM (Invitrogen) was added to each well. Cells wereincubated in transfection mixture for 6 hr and further cultured inantibiotic-free DMEM. Cells were treated under the same conditionswithout siRNA for mock experiments. At various time intervals, thetransfected cells were washed twice with phosphate-buffered saline (PBS,Invitrogen), flash frozen in liquid nitrogen, and stored at −80° C. forreporter gene assays.

In improved dual fluorescence reporter assay, EGFP-C1 encoded enhancedgreen fluorescence protein (GFP), while DsRed2-N1 encoded redfluorescence protein (RFP2). Cells were lysed in ice-cold reporter lysisbuffer (Promega) containing protease inhibitor (complete, EDTA-free, 1tablet/10 ml buffer, Roche Molecular Biochemicals). After clearing theresulting lysates by centrifugation, protein in the clear lysate wasquantified by Dc protein assay kit (Bio-Rad). 240 μg of total celllysate in 160 μl reporter lysis buffer was measured by fluorescencespectrophotometry (Photo Technology International). The slit widths wereset at 4 nm for both excitation and emission. All experiments werecarried out at room temperature. Fluorescence of GFP in cell lysates wasdetected by exciting at 488 nm and recording from 498-650 nm. Thespectrum peak at 507 nm represents the fluorescence intensity of GFP.Fluorescence of RFP2 in the same cell lysates was detected by excitingat 568 nm and recording from 588 nm-650 nm. The spectrum peak at 583 nmrepresents the fluorescence intensity of RFP2. The fluorescenceintensity ratio of target (EGFP) to control (RFP2) fluorophore wasdetermined in the presence of siRNA duplex and normalized to thatobserved in the mocked treated cells. Normalized ratios less than 1.0indicates specific interference.

Western Blotting

Cell lysates were prepared from siRNA-treated cells and analyzed asdescribed above. Proteins in 30 μg of total cell lysate were resolved by10% SDS-PAGE, transferred onto a polyvinylidene difluoride membrane(PVDF membrane, Bio-Rad), and immunoblotted with antibodies against EGFPand DsRed1-N1 (Clontech). For loading control, the same membrane wasalso blotted with anti-actin actibody (Santa Cruz). Protein content wasvisualized with a BM Chemiluminescence Blotting Kit (Roche MolecularBiochemicals). The blots were exposed to x-ray film (Kodak MR-1) forvarious times (between 30 s and 5 min).

Psoralen Photocross-Link of siRNA Duplex

40 μg of siRNA duplex was incubated with 132 μM of a psoralenderivative, 4′-hydroxymethyl-4,5′,8-trimethylpsoralen (HMT) in 200 μlDEPC-treated water at 30° C. for 30 min. Mixtures of siRNA duplex andHMT were exposed to UV 360 nm at 4° C. for 20 min, then denatured bymixing with 400 μl formamide/formaldehyde (12.5:4.5) RNA loading bufferand heating at 95° C. for 15 min. Cross-linked siRNA duplex andnoncross-linked siRNA were resolved by 20% PAGE containing 7M urea inTris-borate-EDTA. Cross-linked siRNA duplexes appeared as a populationwith retarded electrophoretic mobility compared to the noncross-linkedspecies. RNAs were cut from the gel and purified by C18 reverse phasecolumn chromatography (Waters). Purified cross-linked dsRNA andnoncross-linked dsRNA were used in dual fluorescence reporter assays asdescribed above, except that all procedures were performed in the darkto avoid light effects on psoralen. To ensure that the cross-linkdepended on the presence of psoralen, part of the UV 360 nm-treatedmixture was also subjected to UV 254 nm at 4° C. for 20 min.Photoreverse-cross-linked siRNA migrated in 20% polyacrylamide-7 M ureagels with similar mobility to the siRNA duplex without HMT treatment.

Biotin Pull Out Assay for siRNA Isolation from Human Cells

Antisense strands of the siRNA duplex were chemically synthesized andbiotin-conjugated at the 3′ end (Dharmacon, Lafayette, Colo.). Syntheticoligonucleotides were deprotected and annealed with the unmodified sensestrand RNA to form duplex siRNA (ss/as3′-Biotin). HeLa cells, which hadbeen plated at 70% confluency in 100 mm dishes, were cotransfected withduplex siRNA (˜600 pmole) and EGFP-C1 plasmid (1 μg) by alipofectamine-mediated method as described above. At various times, thetransfected cells were washed twice with PBS (Invitrogen) and flashfrozen in liquid nitrogen. Low molecular weight RNA was isolated fromthe cells using a Qiagen RNA/DNA mini kit. Biotinylated siRNA was pulledout by incubating purified RNA with streptavidin-magnetic beads (60 μl)in TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA) containing 1 M NaCl atroom temperature for 3 h. The beads were washed 4 times with 200 μl TEbuffer, resuspended in 100 μl TE buffer and split into two equalaliquots. To one aliquot (50 μl), we added 50 units of shrimp alkalinephosphatase (SAP, Roche Molecular Biochemicals) in 1×SAP buffer andincubated at 37° C. for 1 h. The SAP reaction was then stopped byheating at 65° C. for 15 min and washed 4 times with 200 μl TE buffer.The other aliquot was not treated with SAP. Aliquots of beads with orwithout SAP treatment were incubated with 30 units T4 polynucleotidekinase (T4 PNK, Roche Molecular Biochemicals) in 30 μl 1×PNK buffercontaining 0.2 mCi γ-³²P ATP at 37° C. for 1 h. RNA products wereresolved on 20% polyacrylamide-7M urea gels and ³²P-labeled RNAs weredetected by phosphorimaging.

Study of Duplex siRNA Stability in HeLa Cell Lysate

Unmodified or modified EGFP antisense strand siRNA were 5′-labeled with[gamma-32P] ATP (3000 ci/mM, ICN) by T4 polynucleotide kinases (NewEngland Biolabs) at 37 C for 1 h and chase-kinased by adding 1 mM ATP at37 C for 15 min. Free ATP and Kinase enzyme were removed by Qiagennucleotide removal kit. Duplex siRNA were formed by annealing equalmolar ratio of unmodified or modified sense strand siRNA with the 5′-32Plabeled antisese strand. Duplex formation was confirmed by 20%polyacrylamide gel under native condition. 50 pmole duplex siRNA whichlabeled at 5′ end of the antisense strand were incubated with 500 ugHeLa cytoplasmic extract in 50 ul reaction mixture containing 20 mMHepes, pH 7.9, 100 mM KCl, 10 mM NaCl, 2 mM MgCl2, 10% glycerol. Atvarious time points, 8 μl aliquots were mixed with 16 μl loading buffer(0.01% bromophenol blue, 0.01% xylene cyanol, 98% formaldehyde and 5 mMEDTA). The products were then denatured by heating at 95 C for 10 minand analyzed on 20% polyacrylamide gel containing 7M Urea followed byphosphorimage analysis (Fugi).

Preparation of HeLa cell cytoplasmic extract

HeLa cell cytoplasmic extract was prepared following the Dignam protocolfor isolation of HeLa cell nuclei (Dignam et al., 1983). The cytoplasmicfraction was dialysed against cytoplasmic extract buffer (20 mM Hepes,pH 7.9, 100 mM KCl, 200 μM EDTA, 500 μM DTT, 500 μM PMSF, 2 mM MgCl₂ 10%glycerol). The extract was stored frozen at ˜70° C. after quick-freezingin liquid nitrogen. The protein concentration of HeLa cytoplasmicextract varied between 4 to 5 mg/ml as determined by using a BioRadprotein assay kit.

Preparation of Cap-Labeled Target RNA

For mapping of the target RNA cleavage, a 124 nucleotide transcript wasgenerated corresponding to the EGFP between positions 195 and 297relative to the start codon followed by the 21 nucleotide complement ofthe SP6 promoter sequence. The 124 nucleotide transcript was amplifiedfrom template EGFP-C1 by PCR using the 5′ primer,GCCTAATACGACTCACTATAGGACCTACGGCGTGCAGTGC (SEQ ID NO:33) (T7 promoterunderlined), and the 3′ primer, TTGATTTAGGTGACACTATAGATGGTGCGCTCCTGGACGT(SEQ ID NO:34) (SP6 promoter underlined). The his-tagged mammaliancapping enzyme was expressed in E. coli from a plasmid generouslyprovided by Dr. Stewart Shuman and was purified to homogeneity. Guanylyltransferase labeling was performed by incubating 1 nmole transcript with100 pmole his-tagged mammalian capping enzyme in a 100 μl cappingreaction containing 50 mM Tris-HCl (pH 8.0), 5 mM DTT, 2.5 mM MgCl₂, 1U/l RNasin Rnased inhibitor (promega) and [α-³²P]GTP at 37° C. for 1 hr.The reaction was chased for 30 minutes by supplementing with unlabeledGTP to a concentration of 100 μM. Cap-labeled target RNA was resolved ona 10% polyacrylamide-7M urea gel and was purified.

In Vitro Target RNA Cleavage Assay

siRNA-mediated target RNA cleavage in human cytoplasmic extract wasperformed as described (Martinze et al., 2000, Cell 110-563) with somemodifications. Cap-labeled target RNA of 124 nt was generated as setforth above. siRNA duplex was preincubated with HeLa cytoplasmic extractfor 15 minutes at 37° C. prior to addition of cap-labeled target RNA.After addition of all components, final concentrations were 100 nMsiRNA, 10 nM target RNA, 1 mM ATP, 0.2 mM GTP, 1 UVRNasin, 30 μg/mlcreatine kinase, 25 mM creatine phosphate, and 50% 5100 extract. Thecleavage reactions were further incubated for 1.5 hours and then stoppedby the addition of 8 volumes of proteinase K buffer (200 nM Tris-HCl [pH7.5], 25 mM EDTA, 300 mM NaCl, and 2% w/v SDS). Proteinase K (dissolvedin 50 mM Tris-HCl [pH 8.0], 5 mM CaCl₂, and 50% glycerol) was added to afinal concentration of 0.6 mg/ml. Reactions were extracted withphenol/chloroform/isoamyl alcohol (25:24:1) followed by chloroformalone, and RNA was precipitated with three volumes of ethanol. Sampleswere separated on 8% polyacrylamide-7M Urea gels.

Example XX: Specific Silencing of P-TEFb Expression by siRNA in HeLaCells

RNAi was used to inhibit hCycT1 and CDK9 expression in cultured human(HeLa) cell lines. The short interfering RNA (siRNA) sequence targetinghCycT1 was from position 347 to 367 relative to the start codon, and theCDK9 siRNA sequence was from position 258 to 278 relative to the startcodon. Using lipofectamine, HeLa cells were transfected with hCycT1 orCDK9 siRNA duplex, targeting either hCycT1 or CDK9. To analyze RNAieffects, lysates were prepared from siRNA duplex-treated cells atvarious times after transfection. Western blot experiments were carriedout using anti-hCycT1 and anti-CDK9 antibodies. Briefly, HeLa cells weretransfected with double-stranded (ds) siRNAs targeting RFP, hCycT1, orCDK9. Cells were also transfected with mutant siRNAs (hCycT1 mismatch orCDK9 mismatch) having 2 nucleotide mismatches between the target mRNAand the antisense strand of siRNA at the hypothetical cleavage site ofthe mRNA. Cells were harvested at various times post transfection, theirprotein content resolved on 10% SDS-PAGE, transferred onto PVDFmembranes, and immunoblotted with antibodies against hCycT1 and CDK9.Analysis of immunoblotting experiments reveals that the siRNA targetinghCycT1 inhibited hCycT1 protein expression. siRNA targeting CDK9 wassimilarly specific against CDK9 expression. This RNAi effect depended onthe presence of a 21-nt duplex siRNA harboring a sequence complementaryto the target mRNA, but not on single stranded antisense strand siRNAsnor on an unrelated control siRNA, which targeted a coral (Discosomaspp.)-derived red fluorescent protein (RFP). As a specificity control,cells were also transfected with mutant siRNAs (mismatched siRNA) ofhCycT1 or CDK9, which have two nucleotide mismatches between the targetmRNA and the antisense strand of siRNA at the putative cleavage site ofthe mRNA. Mutant siRNAs showed no interference activity, indicating thespecificity of the RNAi effect. Thus, the siRNAs of the presentinvention specifically silence the subunits of P-TEFb in HeLa cells.

Example XXI: Specific Silencing of P-TEFb by siRNA at the mRNA Level andStability of CDK9

To determine the specificity of P-TEFb knockdown by siRNA at the mRNAlevel, RT-PCR was performed to reveal the effect of siRNA on the levelof mRNA involved in P-TEFb expression. Briefly, HeLa cells weretransfected with hCycT1 ds siRNA and CDK9 ds siRNA, harvested at varioustimes after transfection and mRNAs extracted. One-step RT-PCR wasperformed, setting the specific primer for hCycT1 and CDK9amplification. RT-PCR products were resolved in 1% agarose gel andviewed by ethidium bromide staining. Transfection of cells with siRNAduplex targeting hCycT1 (hCycT1 ds) significantly reduced hCycT1expression, but had no effect on CDK9 mRNA.

On the other hand, transfection of cells with siRNA duplex targeted toCDK9 (CDK9 ds) significantly interfered with the expression of CDK9, butnot hCycT1. These results suggested that hCycT1 knockdown did not resultin decreased transcription of CDK9 mRNA. The siRNA duplex started tocause an RNAi effect as early as 6-18 hours post transfection andgradually increased with time, peaking at 30 h, and decreased between54-66 h. The time-dependent effect of siRNA indicates that siRNAs needto be processed or assembled into an active complex with cellularfactors for effective RNA interference. A time lag was also seen betweenthe degradation of target mRNA (starting at 6 hours post siRNAtransfection, as shown by semi-quantitative RT-PCR) and the half-life ofthe existing protein expressed by the target gene, because proteinlevels did not show any down-regulation until 18-30 hours post siRNAtransfection. Combined with Western blot analysis, semi-quantitativeRT-PCR not only confirms the specific knockdown of P-TEFb by siRNA atthe mRNA level, but also suggests that forming a complex with hCycT1 isa prerequisite for maintaining the stability of CDK9 proteins in livingcells. Thus, hCycT1 siRNA down-regulated hCycT1 levels by the RNAipathway, while down-regulating CDK9 levels by promoting its degradationwithout affecting its gene expression at the mRNA level. This indicatesthat the use of hCycT1 siRNA, even without added CDK9 siRNA, is able todown regulate both P-TEFb and CDK9 activity.

Example XXII: hCycT1 and CDK9 Knockdown are not Lethal to Human Cells

To analyze the viability of cells subjected to P-TEFb gene silencing, apEGFP-C1 reporter plasmid, harboring enhanced green fluorescent protein[GFP] under the cytomegalovirus (CMV) immediate early promoter, plushCycT1 and CDK9 siRNAs were co-transfected into HeLa cells usinglipofectamine. Briefly, HeLa cells were cotransfected by Lipofectamine™with pEGFP-C1 reporter (GFP) plasmid and siRNAs. Four siRNA duplexes,including a control duplex targeting RFP and three duplexes targetinghCycT1, CDK9, and CDK7, were used in these experiments. Reporter geneexpression was monitored at 50 hours post transfection by fluorescenceimaging in living cells. Cellular shape and density were recorded byphase contrast microscopy. Reporter gene (GFP) expression, driven bycytomegalovirus (CMV) immediate early promoter, was monitored in livingcells. Cellular morphology and density were monitored by phase contrastmicroscopy. GFP expression was not affected by hCycT1 or CDK9 knockdown.Cells with P-TEFb knockdown had normal shape and growth rate. At 50hours post transfection, cell density reached ˜90% to 100% confluency.

For comparison, cells were transfected with siRNA targeting CDK7, awell-characterized kinase required for TFIIH, an essential transcriptionfactor, to phosphorylate the CTD of RNA pol II at the step of promoterclearance during initiation of transcription. Kin28, a protein inSaccharomyces cerevisiae that is equivalent to CDK7 in mammals, is anessential gene product that phosphorylates Ser5 of the CTD YSPTSPSrepeat region (Komarnitsky et al. (2000), Genes Dev., 14, 2452-2460;Rodriguez et al. (2000), Mol. Cell. Biol., 20, 104-112; Schroeder et al.(2000), Genes & Dev., 14, 2435-2440) and is required to recruit the mRNAcapping enzyme to the transcription machinery (Cho et al. (1997), Genes& Dev., 11, 3319-3326; McCracken et al. (1997), Genes & Dev., 11,3306-3318; McCracken et al. (1997), Nature, 385, 357-361; Yue et al.(1997), Proc. Natl. Acad. Sci. USA, 94, 12898-12903). CDK7 is abifunctional enzyme in larger eukaryotes, promoting both CDK activationand transcription (Harper and Elledge. (1998), Genes & Dev., 12,285-289). As expected, reduction of CDK7 levels by RNAi led to a lowerreporter (GFP) expression and an arrest in cellular growth (FIG. 4,panel d). CDK7 knockdown cells were smaller than control cells andshowed blebbing (FIG. 4, panel h), indicating that unlike RNAi ofP-TEFb, CDK7 gene silencing had an adverse affect on transcription, cellmorphology and cell growth.

Cellular viability was next analyzed under various siRNA treatments. Atvarious times after transfection, cell viability was assessed by trypanblue exclusion (see below). Briefly, HeLa cells were cotransfected byLipofectamine™ with pEGFP-C1 reporter (GFP) plasmid and siRNAs (seeExperimental Procedures). Four siRNA duplexes, including a controlunrelated duplex and three duplexes targeting hCycT1, CDK9, and CDK7,were used in these experiments. At various times after transfection,cells floating in the medium were collected and counted in the presenceof 0.2% trypan blue (see Experimental Procedures). Cells that took updye (stained blue) were not viable. Over a 66 hours time courseexperiment, the rate of cell death in P-TEFb (hCycT1 or CDK9) knockdowncells was comparable to that in control cells with unrelated siRNAtreatment, while CDK7 knockdown cells showed a significant increase incell death. These results indicate that P-TEFb knockdown is not lethalto human cells, while a much more stringent threshold for CDK7 isrequired to maintain cell viability and growth.

Example XXIII: hCycT1 and CDK9 RNAi Inhibit HIV-1 Tat Transactivation inHuman Cells

A dominant paradigm for Tat up-regulation of HIV gene expression at thelevel of transcription elongation revolves around the ability of theTat-TAR RNA complex to bind to P-TEFb and stimulate phosphorylation ofthe CTD and Spt5, thereby overriding the elongation arrest elicited byDSIF and NELF (Ping and Rana, 2001, supra; Price, 2000, supra). To testwhether siRNAs that targeted sequence elements of P-TEFb wouldspecifically block Tat transactivation, Magi cells were cotransfectedwith the Tat expression construct pTat-RFP and hCycT1 or CDK9 ds siRNAor as controls, antisensehCycT1 or CDK9 siRNA, mutant hCycT1 or CDK9siRNA, or non-P-TEFb duplex siRNA. Magi, a HeLa cell line harboring asingle copy of persistently transfected HIV-1 LTR-β-galactosidase gene,is programmed to express the CD4 receptor and the CCR5 coreceptor forHIV-1, making them a model cell line for measuring HIV replication(Kimpton and Emerman, 1992, supra). It was confirmed that the HIV-1Tat-RFP fusion protein was expressed under control of the CMV earlypromoter in all transfected cells by Western blot, using anti-RFPantibody.

Tat-RFP strongly enhanced β-galactosidase gene expression, which isunder control of the HIV-1 LTR promoter in transfected Magi cells. Tattransactivation was determined by calculating the ratio ofβ-galactosidase activity in pTat-RFP transfected cells to the activityin cells without pTat-RFP treatment. Inhibitory activity was determinedby normalizing Tat-transactivation activity to the amount of Tat-RFPprotein (represented by RFP fluorescence intensity as described inExperimental Procedures) in the presence and absence of siRNA. Briefly,twenty-four hours after pre-treating Magi cells with siRNA, they werecotransfected with pTat-RFP plasmid and various siRNAs. Cells wereharvested 48 h post pTat-RFP transfection, and activity □□ ofβ-galactosidase in clear cell lysates was measured (see ExperimentalProcedures). Magi cells were cotransfected with ds siRNAs targetinghCycT1 and CDK9, with antisense (as) RNA strands, or mutant (mm) siRNAs.GFP ds siRNA was used as an unrelated control siRNA, while Tat ds siRNA,targeting the mRNA encoding Tat sequence, was used as a positivecontrol. Means±SD of two experiments are shown. Under standardexperimental conditions, Tat-RFP enhanced gene transactivation 20- to25-fold. This activation was strongly inhibited by cotransfecting hostMagi cells with the specific ds siRNAs targeting hCycT1 and CDK9, butnot with antisense (as) RNA strands, mutant (mm) siRNAs or an unrelatedcontrol siRNA.

Specific RNA interference with hCycT1 and CDK9 expression in Magi cellswas demonstrated by Western blot analysis. Briefly, Magi cells wereco-transfected with pTat-RFP plasmid and various siRNAs. Cells wereharvested at 48 hours post transfection, resolved on 10% SDS-PAGE,transferred onto PVDF membranes, and immunoblotted with antibodiesagainst hCycT1 and CDK9. RNAi activities in Magi cells treated withantisense (as) strands of hCycT1 and CDK9 siRNAs, cells treated with dssiRNA targeting hCycT1 and CDK9, cells treated with mutant hCycT1 siRNA(hCycT1 mm) or mutant CDK9 siRNA (CDK9 mm) were examined. GFP ds siRNAwas used as an unrelated control, while Tat ds RNAi was used to targetmRNA encoding Tat. The inhibition of Tat transactivation correlated wellwith the knockdown of hCycT1 and CDK9 protein levels by the hCycT1 andCDK9 siRNAs. Syncytia formation and LTR activation were reduced inhCycT1 ds siRNA-treated cells. From these results, it can be concludedthat siRNA targeting P-TEFb can inhibit Tat-transactivation in humancells without affecting cellular viability, thus making siRNA targetingP-TEFb an excellent candidate for treatment of patients infected withHIV.

Example XXIV: hCycT1 and CDK9 RNAi Inhibit HIV-1 Infectivity

The next question addressed was whether targeting the human P-TEFbcomplex by RNAi inhibited HIV replication. To investigate this question,HeLa-CD4-LTR/β-galactosidase (Magi) cells were transfected withhomologous and mismatched siRNAs directed against hCycT1 or CDK9 and 16hours later infected the Magi cells with various concentrations ofHIV_(NL-GFP), an infectious molecular clone of HIV-1. HIV-1 Tat-mediatedtransactivation of the LTR led to β-galactosidase production that wasquantified 36 hours post-infection. Briefly, LTRM-galactosidase (Magi)cells transfected with homologous and mismatched siRNAs directed againstCycT1 or CDK9. Cells were also mock transfected without siRNA ortransfected with an unrelated ds siRNA against the RFP sequence. Sixteenhours later, cells were infected with NL-GFP, an infectious molecularclone of HIV-1. Cells infected with virus and not treated witholigofectamine were examined. HIV-1 Tat-mediated transactivation of theLTR led to ν-galactosidase production, which was quantified 36 hourspost-infection. Cells treated with ds siRNA targeting GFP-Nef andtargeting the mRNA encoding Tat sequence served as positive controls.These controls previously showed decreased levels of β-galactosidaseactivity and viral infectivity (Jacque et al. 2002 Nature 418:435-8).

ds siRNA directed against hCycT1 or CDK9 inhibited viral infectivity.Doubling dilutions of the inoculums are consistent with an 8-folddecrease in viral infectivity. Control experiments using siRNA duplexescontaining mismatched sequences (see Experimental Procedures) and anunrelated ds siRNA against the RFP sequence showed no antiviralactivities. Consistent with our previous results (Jacque et al., 2002,supra), siRNA targeting GFP-Nef and Tat led to an 8-fold decrease inviral infectivity. No significant toxicity or cell death was observedduring these experiments, suggesting further that P-TEFb knockdown wasnot lethal. These results demonstrate that HIV infectivity can bemodulated by siRNAs targeting CycT1 or CDK9, both components of P-TEFb,indicating that the use of siRNA targeting either subunit is a viabletreatment for patients with HIV.

Example XXV: Method of Treating Cancer by Inhibiting P-TEFb

An intriguing finding is that genes linked to embryonic development andshowing down-regulation in P-TEFb knockdown cells (as described above)also participate in tumorogenesis and metastasis. Dysfunction of proteintyrosine kinases or aberrations in key components of the signalingpathways they activate can lead to severe pathologies such as cancer,diabetes and cardiovascular disease. For example, overexpression of EGFRhas been implicated in mammary carcinomas, squamous carcinomas andglioblastomas (Schlessinger (2002), Cell, 110, 669). AXL, anotherreceptor tyrosine kinase, was originally identified with oncogenicpotential and transforming activity in myeloid leukemia cells (Burchertet al. (1998), Oncogene, 16, 3177-3187). Elevated TGF-beta levels cancontribute to tumor progression and metastasis (Attisano and Wrana,2002, supra; Massague, 2000, supra). Lysyl oxidase (LOX class II), anextracellular matrix remodeling enzyme, is up-regulated in prostatictumor, cutaneous and uveal cell lines (Kirschmann et al. (2002), CancerRes., 62, 4478-4483). Down-regulating these genes by P-TEFb knockdownusing siRNA targeting CDK9 or CycT1 thus provides a new therapeuticstrategy for inhibiting tumorigenesis and metastasis.

Genes involved in mediating progression through the cell cycle and ascheckpoints in cancer were regulated by P-TEFb. Cyclin G1 is thedownstream target of the P53 pathway and plays a role in G2/M arrest,damage recovery and growth promotion after cellular stress (Kimura etal. (2001), Oncogene, 20, 3290-3300). Cyclin D, a cell-cycle regulatoryprotein essential for G1/S transition, has been identified as apotential transforming gene in lymphoma (Motokura and Arnold (1993),Curr. Opin. Genet. Dev., 3, 5-10). Misregulation of the activity of itspartner, CDK4/6, by overexpression of Cyclin D leads tohyperproliferative defects and tumor progression (Ortega et al. (2002),Biochim. Biophys. Acta, 1602, 73-87). Several marker genes in cancercells (class V) are also regulated by P-TEFb. For example, breastcancer-specific protein 1 (BCSG1) is overexpressed in advanced,infiltrating breast cancer and colorectal tumors (Lu et al. (2001),Oncogene, 20, 5173-5185). Another example is soluble urokinaseplasminogen activator receptor (SUPAR), which is present in highconcentrations in cystic fluid form ovarian cancer, tumor tissue ofprimary breast cancer, and gynecological cancer (Riisbro et al. (2002),Clin. Cancer. Res., 8, 1132-1141; Wahlberg et al. (1998), Cancer Res.,58, 3294-3298). Although the functions of these marker genes are stillunknown, their high correlation with cancer has been used for prognosisin cancer therapy. The down-regulation of cyclin D and cancer markergenes by P-TEFb knockdown offers a method of cancer therapy. Briefly, atherapeutically effective amount of one of more of the pharmaceuticalcompositions of the invention is administered to a patient having adisorder characterized by unwanted or aberrant cellular proliferation asdescribed herein.

Example XXVI: Specific Silencing of P-TEFb In Vivo

The effect of downregulating P-TEFb in vivo is assayed by administeringsiRNA targeted to CDK9 and/or CycT1 in an animal model. Any appropriateanimal model can be used, for example, including but not limited to,rodent cancer models such as those available from the Mouse Models ofHuman Cancers Consortium (MMHCC) Repository (NCI, Frederick, Md.); theOncomouse™ as described in U.S. Pat. Nos. 4,736,866, 5,087,571 and5,925,803 (Taconic); or rodent or non-human primate models of HIVinfection, such as the SCID-hu mouse.

For example, in a mouse model, the siRNA is administered usinghydrodynamic transfection as previously described (McCaffrey, 2002,supra; Liu, 1999, supra), by intravenous injection into the tail vein(Zhang, 1999, supra); or by viral delivery (Xia, 2002, supra). Atvarious time points after administration of the selected siRNA, mRNAlevels for CDK9 and/or CycT1 can be measured. Additionally, the siRNAcan be labeled, and the half-life of the siRNA molecules can be trackedusing methods known in the art. Using electroporation, RNaseIII-prepared siRNA can be delivered into the post-implantation mouseembryos. 0.03:g-0.3:g siRNA can efficiently silence reporter geneexpression in different regions of the neural tube or other cavities ofthe mouse embryo (Calegari (2002), supra). Using rapid injection of thesiRNA-containing physiological solution into the tail vein of postnatalmice, 0.5-5:g siRNA can cause 36±17%-88%±3% inhibition of target geneexpression. The effect of RNAi is siRNA dose-dependent and can persistfor approximately 4 days after siRNA delivery (Lewis (2002), supra). Bydirect injection, 5-40:g siRNA can be used to silencing target geneexpression in the liver, which is central to metabolism (Lewis (2002),supra; McCaffrey (2002), supra).

Any appropriate parameter can be observed to investigate the effect ofP-TEFb expression. For example, changes in gene expression can bedetermined, such as changes in the expression of any one or more of thegenes listed herein. In a mouse cancer model, appropriate parameters caninclude survival rates, tumor growth, metastasis, etc. In a simian HIVmodel, for instance parameters that can be determined include, but arenot limited to, infectivity, viral load, survival rates, and rates andseverity of secondary AIDS-associated illnesses.

Such models may also be useful for evaluating various gene deliverymethods and constructs, to determine those that are the most effective,e.g., have the greatest effect, or have a desirable half-life ortoxicity profile, for instance.

Example XXVII: Specific Silencing of hSpt5 Expression by siRNA in HeLaCells

To inhibit hSpt5 expression in a cultured human cell line using RNAi,siRNA targeting an hSpt5 sequence from position 407 to 427 relative tothe start codon was designed. Magi cells were then transfected withhSpt5 duplex siRNA using Lipofectamine (Invitrogen). To evaluate theeffects of hSpt5 RNAi, total cell lysates were prepared fromsiRNA-treated cells harvested at various time points after transfection.hSpt5 mRNA or protein levels were then analyzed by RT-PCR or westernblot using anti-hSpt5 antibodies, respectively. These experiments showedthat cells transfected hSpt5 siRNA had significantly lowered hSpt5 mRNAand protein expression, indicating that RNAi of hSpt5 had occurredsuccessfully. This knockdown effect was dependent on the presence of a21-nt siRNA duplex harboring a sequence complementary to the mRNAtarget. Mock-treated (no siRNA), single-stranded antisense hSpt5 siRNA,mismatched hSpt5 duplex siRNA, containing two nucleotide mismatchesbetween the target mRNA and siRNA antisense strand at the putativecleavage site of the target mRNA did not affect hSpt5 mRNA or proteinslevels. This suggested that hSpt5 knockdown was specific to duplex siRNAexactly complementary to the hSpt5 mRNA target. In evaluating eithermRNA or protein levels, human Cyclin T1 (hCycT1) was used as an internalcontrol, showing that the effects of hSpt5 siRNA were specific to hSpt5and did not effect hCycT1 mRNA or protein levels. Taken together, theseresults suggested that hSpt5 knockdown was sequence specific and led tosignificantly decreased hSpt5 mRNA and proteins levels.

Example XXVIII: Specific Silencing of Spt5 by siRNA at the mRNA Level

To determine the specificity of Spt5 knockdown by siRNA at the mRNAlevel, RT-PCR is used to reveal the effect of siRNA on the level of mRNAinvolved in Spt5 expression. Briefly, HeLa cells are transfected withSpt5 ds siRNA, harvested at various times after transfection and mRNAsare extracted. One-step RT-PCR is performed, using specific primers forSpt5 amplification. A control is run concurrently using primers specificfor another, unrelated gene, e.g., CDK9, CycT1, or actin. RT-PCRproducts are resolved in 1% agarose gel and viewed by ethidium bromidestaining. Changes in Spt5 mRNA levels with time, while the levels ofmRNA of the unrelated gene remain unaltered, indicate that the effect ofthe siRNA is specific.

Example XXIX: Viability of Human Cells with Spt5 Knockdown

Cellular viability under various siRNA treatments was analyzed by trypanblue exclusion. Knowing that the kinetics of hSpt5 peaked at 42-54 hpost-transfection, the viability of cells during an hSpt5 knockdown timecourse experiment could be evaluated. Cell viability was assessed usingtrypan blue exclusion at various times after transfection of varioussiRNAs. During the 66 h time course experiment, the number of non-viablehSpt5 knockdown cells observed was comparable to mock-treated cells.Cells transfected with single-stranded antisense hSpt5 siRNA ormismatched hSpt5 duplex siRNA that did not show hSpt5 knockdown alsoshowed minimal changes in cell viability. The positive control for thisexperiment was human capping enzyme (HCE), which is a bifunctionaltriphophsatase-guanylyltransferase required for capping mRNA (reviewedin Bentley et al., 2002 Curr Opin Cell Biol 14:336-342). HCE is verylikely to be essential for cell viability as the HCE homolog cel-1 in C.elegans is essential (Srinivasan et al., 2003 J Biol Chem278:14168-14173). In contrast to hSpt5 knockdown cells, HCE knockdowncells showed a significant increase in cell death over the course of theknockdown experiment. These results indicated that hSpt5 knockdown wasnot lethal to human cells, while a much more stringent requirement forHCE expression was essential for cell viability.

Cell viability in vivo under siRNA treatment can also be evaluated byfluorescence imaging. pEGFP-C1 reporter plasmid (harboring enhancedgreen fluorescent protein [GFP]) and siRNAs are cotransfected into HeLacells using Lipofectamine™. Briefly, HeLa cells are cotransfected byLipofectamine™ with pEGFP-C1 reporter (GFP) plasmid and siRNAs. Ingeneral, four siRNA duplexes, including a control duplex targeting RFPand duplexes targeting Spt5 are used in these experiments. Reporter geneexpression is monitored at 50 hours post transfection by fluorescenceimaging in living cells. Cellular shape and density are recorded byphase contrast microscopy.

Example XXX: hSpt5 RNAi Inhibits HIV-1 Tat Transactivation in HumanCells

A dominant paradigm for Tat up-regulation of HIV gene expression at thelevel of transcription elongation revolves around the ability of theTat-TAR RNA complex to bind to P-TEFb and stimulate phosphorylation ofthe CTD and Spt5, thereby overriding the elongation arrest elicited byDSIF and NELF (Ping and Rana (2001), supra; Price (2000), supra).

To examine whether hSpt5 was required for HIV-1 Tat transactivation invivo, Tat transactivation during hSpt5 knockdown in Magi cells wasmonitored. Magi cells are a HeLa cell line harboring a stably integratedsingle copy of the HIV-1 5′ LTR-β-galactosidase gene. These cells arealso genetically programmed to express the CD4 receptor as well as CCR5coreceptor for HIV-1 infection (Kimpton and Emerman, 1992 J Virol66:2232-2239); see below). In this experiment, Magi cells wereco-transfected with Tat expression plasmid pTat-RFP and hSpt5 duplexsiRNA. Co-transfection with Tat siRNA was used as a positive control forinhibition of Tat transactivation while single-stranded antisense hSpt5siRNA and mismatched siRNA were used as negative controls. Tattransactivation and protein levels were evaluated by harvesting cells 48h post transfection, which was within the timeframe that hSpt5 knockdownpeaked. Expression of HIV-1 Tat-RFP under the control of the CMV earlypromoter was confirmed by western blot using anti-RFP antibody and RFPfluorescence measurement on a fluorescence spectrophotometer (data notshown). In addition, immunoblot analysis confirmed that hSpt5 siRNAspecifically inhibited hSpt5 protein expression in the absence andpresence of HIV-1 Tat protein in Magi cells (data not shown).

Tat-RFP enhances the expression of genes that are under the control ofthe HIV-1 5′ LTR promoter. In this experiment, Tat transactivation wasmeasured by assaying the β-galactosidase activity resulting fromexpression of the β-galactosidase gene under the HIV-1 5′ LTR promoter.To quantify the effects of various siRNAs on HIV-1 Tat transactivation,the ratio between β-galactosidase activity in cells transfected withpTat-RFP (with or without siRNAs) and mock-treated cells not transfectedwith pTat-RFP was determined. In Magi cells, Tat-RFP strongly stimulatesthe expression of β-galactosidase, represented by a 13-fold increase inTat transactivation. On the other hand, Tat transactivation was stronglyinhibited in cells transfected with Tat siRNA, as previously shown(Surabhi and Gaynor 2002 J Virol 76:12963-12973). Tat transactivationwas similarly inhibited when cells were transfected with hSpt5 duplexsiRNA, exhibiting only ˜30% of the Tat transactivation observed withTat-RFP alone. Neither antisense hSpt5 siRNA nor mismatched hSpt5 siRNAshowed any effect on Tat transactivation. These results indicated hSpt5knockdown caused by siRNA specifically targeting hSpt5 mRNA inhibitedHIV-1 Tat transactivation in human cells. These results stronglysupported an important role for hSpt5 in Tat transactivation in vivo andsuggested that RNAi of hSpt5 had the potential to inhibit HIV-1replication.

Example XXXI: hSpt5 siRNAs Inhibit hSpt5 Protein Expression in thePresence or Absence of Tat Expression

Specific RNA interference with Spt5 expression in Magi cells wasdemonstrated by Western blot analysis. Briefly, Magi cells wereco-transfected with pTat-RFP plasmid and various siRNAs. Cells wereharvested at 48 hours post-transfection, resolved on 10% SDS-PAGE,transferred onto PVDF membranes, and immunoblotted with antibodiesagainst Spt5 or hCycT1. RNAi activities in Magi cells treated withantisense (AS) strands of Spt5 siRNAs and in cells treated with ds siRNAtargeting Spt5 were examined. RNAi activities in cells treated withmismatch Spt5 (hCycT1 mm) siRNAs with two mismatches were also examined.From the results, it can be concluded that siRNA targeting hSpt5 caninhibit hSpt5 protein expression in the presence or absence of Tatprotein, making siRNA targeting hSpt5 an excellent candidate compoundfor treatment of patients infected with HIV.

Example XXXII: RNAi Inhibition of HIV-1 Infectivity

Since hSpt5 knockdown effectively inhibited Tat transactivation, we nextdetermined whether hSpt5 knockdown could inhibit HIV-1 replication. Toevaluate the effect of hSpt5 knockdown on HIV-1 replication, a doublesiRNA transfection protocol was used to maximize the knockdownefficiency of hSpt5 during HIV-1 infection. Magi cells were transfectedwith siRNA directed against hSpt5. Cells mock transfected without siRNA,or transfected with single-stranded antisense hSpt5 siRNA or mismatchhSpt5 siRNA were used as negative controls. Transfection with Nef siRNAwas used as a positive control. 24 h after the first transfection, asecond siRNA transfection was performed. 24 h later, doubly transfectedcells were infected with various concentrations of HIV_(NL-GFP), aninfectious molecular clone of HIV-1. Knockdown of hSpt5 protein levelswas then evaluated 48 h post infection in doubly transfected cells. Aneven larger decrease in hSpt5 protein levels was observed in doublytransfected cells as compared to singly transfected cells, suggestingthat more robust knockdown of gene expression can be achieved using thisdouble transfection method.

HIV-1 Tat-mediated transactivation of the 5′ LTR occurring in cellsinfected with virus led to β-galactosidase production, which was alsoquantified 48 h post-infection. In this single-cycle replication assayfor evaluating HIV-1 replication, β-gal activity reflected the activityof reverse transcriptase and viral replication of varying amounts ofviral inoculum. Therefore, changes in 3-gal activity could be directlycorrelated to changes in the efficacy of HIV replication. The positivesiRNA control targeting HIV Nef showed decreased levels of β-galactivity and viral infectivity, as shown previously (FIG. 32; (Jacque etal., 2002 Nature 418:435-438). Double-stranded siRNA directed againsthSpt5 showed a similar decrease in β-gal activity when compared with Nefknockdown. This observed decrease was equivalent to the β-gal activitymeasured when using 32 times less viral inoculum with mock-treatedcells, indicating that hSpt5 knockdown had significantly reduced HIVreplication. Control experiments using hSpt5 single-stranded antisenseor mismatched duplex siRNA duplexes showed no antiviral activities. Inaddition, no significant toxicity or cell death was observed duringthese experiments, suggesting that hSpt5 knockdown was not lethal evenin the context of HIV-1 infection. These results demonstrated that HIVreplication was modulated by siRNAs targeting hSpt5, furtherestablishing an important role for hSpt5 in Tat transactivation andHIV-1 replication in vivo.

Example XXXIII: Specific Silencing of TEFs In Vivo

The effect of downregulating TEFs in vivo is assayed by administeringsiRNA targeted to one or more TEFs, e.g. Spt4, Spt5, and/or Spt6, in ananimal model. The siRNA is administered using hydrodynamic transfectionas previously described (McCaffrey (2002), supra; Liu (1999), supra), byintravenous injection into the tail vein (Zhang (1999), supra); or byviral delivery (Xia (2002), supra). At various time points afteradministration of the selected siRNA, mRNA levels for one or more TEFs,e.g., Spt4, Spt5, and/or Spt6 are measured. Additionally, the siRNA canbe labeled, and the half life of the siRNA molecules is tracked usingmethods known in the art. Using electroporation, RNase III-preparedsiRNA can be delivered into the post-implantation mouse embryos.0.03:g-0.3:g siRNA can efficiently silence reporter gene expression indifferent regions of the neural tube or other cavities of the mouseembryo (Calegari (2002), supra). Using rapid injection of thesiRNA-containing physiological solution into the tail vein of postnatalmice, 0.5-5:g siRNA can cause 36±17%-88%±3% inhibition of target geneexpression. The effect of RNAi is siRNA dose-dependent and can persistfor approximately 4 days after siRNA delivery (Lewis (2002), supra). Bydirect injection, 5-40:g siRNA can be used to silencing target geneexpression in the liver, which is central to metabolism (Lewis (2002),supra; McCaffrey (2002), supra).

Experimental Procedures for Examples XX-XXXIII

siRNA Preparation

Design of siRNAs Against CDK9/CycT1 The targeted region in the mRNA, andhence the sequence of CycT1 or CDK9-specific siRNA duplexes was designedfollowing the guidelines provided by Dharmacon (Lafayette, Colo.).Briefly, starting 100 bases downstream of the start codon, the first AAdimer was located and the next 19 nucleotides were then recordedfollowing the AA dimer.

Criteria were set such that the guanosine and cytidine content (G/Ccontent) of the AA-N19 21 base-sequence must be less than 70% andgreater than 30%. The search continued downstream until the conditionswere met. The 21-mer sequence was subjected to a BLAST search againstthe human genome/NCBI EST library to ensure only the desired gene wastargeted. The siRNA sequence targeting hCycT1 was from position 347-367relative to the start codon. The siRNA sequence targeting CDK9 was fromposition 258-278 relative to the start codon. siRNA sequences used inour experiments were: hCycT1 ds (5′-UCCCUUCCUGAUACUAGAAdTdT-3′) (SEQ IDNO:3); hCycT1 mm (5′-UCCCUUCCGUAUACUAGAAdTdT-3′) (SEQ ID NO:4); CDK9 ds(5′-CCAAAGCUUCCCCCUAUAAdTdT-3′) (SEQ ID NO:5); CDK9 mm(5′-CCAAAGCUCUCCCCUAUAAdTdT-3′) (SEQ ID NO:6); CDK7 ds(5′-UUGGUCUCCUUGAUGCUUUdTdT-3′) (SEQ ID NO:17); Tat ds(5′-GAAACGUAGACAGCGCAGAdTdT-3′) (SEQ ID NO:18); GFP ds(5′-GCAGCACGACUUCUUCAAGdTdT-3′) (SEQ ID NO:19); and RFP ds(5′-GUGGGAGCGCGUGAUGAACdTdT-3′) (SEQ ID NO:20). Underlined residuesrepresent the mismatched sequence to their targets.

hCycT1 contains an amino-terminal cyclin box motif (amino acids 1-298)that is conserved in the cyclin type protein family, a putativecoiled-coil motif (amino acids 379-430) and a histidine-rich motif(amino acids 506-530). The hCycT1 sequence containing amino acids 1-303is sufficient to form complexes with Tat-TAR and CDK9, as CDK9 binds tothe cyclin box (amino acids 1-250) of CycT1. A Tat:TAR recognition motif(TRM) in the hCycT1 sequence that spans amino acids 251-272 is necessaryfor forming complex with Tat and TAR. Residues 252-260 of hCycT1 havebeen demonstrated to interact with the TAR RNA loop, suggesting thatamino acids 261-272 are involved in interaction with Tat core domain. Acritical cysteine (amino acids 261) has been identified as a absolutelyrequiring residue for the Tat and hCycT1 interaction. The targetedregion in the mRNA and hence the sequence of hCycT1-specific siRNAduplexes can be designed targeting to the Cyclin box region or theregion for Tat-TAR interaction. Using the guidelines provided byDharmacon (Lafayette, Colo.) as discussed above, other potential siRNAtarget sequences include the following: relative to the start codon, thesiRNA sequences targeting hCycT1 can be from position 238-278, 502-522,758-778, 769-789 etc. Based on the guidelines of Dharmacon as discussedabove, additional siRNA sequences suitable for targeting CDK9 can befrom position 220-240, 258-278, 379-399 relative to the start codon.

Design of siRNAs Targeting Spt5

The targeted region in the mRNA, and hence the sequence of Spt5-specificsiRNA duplexes, was designed following the guidelines provided byDharmacon (Lafayette, Colo.). Briefly, beginning 100 bases downstream ofthe start codon, the first AA dimer was located and then the next 19nucleotides following the AA dimer were recorded. Ideally, the guanosineand cytidine content (G/C content) of the AA-N19 21 base-sequence wouldbe less than 70% and greater than 30%. The search was continueddownstream until the conditions were met. The 21-mer sequence wassubjected to a BLAST search against the human genome/NCBI EST library toensure only the desired gene was targeted. The siRNA sequence targetinghSpt5 was from position 407-427 relative to the start codon. siRNAsequences used in the experiments described herein were: hSpt5ds(5′-AACTGGGCGAGTATTACATGAdTdT-3′) (SEQ ID NO: 8); h Spt5 mm(5′-AACTGGGCGGATATTACATGAdTdT-3′) (SEQ ID NO: 9); Tat ds(5′-GAAACGUAGACAGCGCAGAdTdT-3′) (SEQ ID NO: 18); GFP ds(5′-GCAGCACGACUUCUUCAAGdTdT-3′) (SEQ ID NO: 19); and RFP ds(5′-GUGGGAGCGCGUGAUGAACdTdT-3′) (SEQ ID NO: 20). Underlined residuesrepresent the sequences mismatched to their targets.

Using the guidelines provided by Dharmacon (Lafayette, Colo.) asdiscussed above, other potential siRNA sequences targeting Spt5, as wellas siRNA sequences targeting Spt4 or Spt6, can be identified.

SiRNA Synthesis and Maintenance

21-nt RNAs were chemically synthesized as 2′ bis(acetoxyethoxy)-methylether-protected oligos by Dharmacon (Lafayette, Colo.). Syntheticoligonucleotides were deprotected, annealed to form dsRNAs and purifiedaccording to the manufacturer's recommendation. Successful duplexformation was confirmed by 20% non-denaturing polyacrylamide gelelectrophoresis (PAGE). All siRNAs were stored in DEPC (0.1% diethylpyrocarbonate)-treated water at −80° C.

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EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

What is claimed:
 1. A small interfering RNA (siRNA), comprising a sensestrand and an antisense strand wherein the antisense strand comprises atleast one internal 2′-fluoro ribonucleotide modification, at least oneinternal 2′-deoxy ribonucleotide modification, and a sequencesufficiently complementary to a single target mRNA sequence to directtarget-specific RNA interference (RNAi), wherein one or more purinenucleotides in one or both of the sense or antisense strands ischemically modified with a 2′-sugar modification and wherein in vivostability is enhanced as compared to a corresponding unmodified siRNA orwherein target efficiency is enhanced compared to a correspondingunmodified siRNA.
 2. The siRNA of claim 1 which is sufficientlycomplementary to a target mRNA, said target mRNA specifying the aminoacid sequence of a cellular protein or the amino acid sequence of aviral protein.
 3. The siRNA of claim 1, further comprising at least oneadditional modified nucleotide selected from the group consisting of (a)a sugar-modified nucleotide, (b) a nucleobase-modified nucleotide, (c) a2′-deoxy ribonucleotide that is present within the sense strand, (d) a2′-fluoro modified ribonucleotide that is present within the sensestrand, (e) a modified nucleotide selected from the group consisting ofa 2′-fluoro, 2′-amino and 2′-thio modified ribonucleotide, (f) modifiednucleotides which are one or both of a 2′-fluoro modified ribonucleotideand a 2′-deoxy ribonucleotide that is present within the sense strand,(g) a modified nucleotide selected from the group consisting of2′-fluoro-cytidine, 2′-fluoro-uridine, 2′-fluoro-adenosine,2′-fluoro-guanosine, 2′-amino-cytidine, 2′-amino-uridine,2′-amino-adenosine, 2′-amino-guanosine and2′-amino-butyryl-pyrene-uridine, (h) a modified nucleotide selected fromthe group consisting of 5-bromo-uridine, 5-iodo-uridine,5-methyl-cytidine, ribo-thymidine, 2-aminopurine, 5-fluoro-cytidine, and5-fluoro-uridine, 2,6-diaminopurine, 4-thio-uridine; and5-amino-allyl-uridine, and (i) a backbone-modified nucleotide.
 4. ThesiRNA of claim 1, wherein the sense strand is crosslinked to theantisense strand, or wherein a 3′ OH terminus of the sense strand orantisense strand is modified.
 5. The siRNA of claim 1, comprising about10 to 50 residues in length, between about 15 to 45residues in length,between about 20 to 40 residues in length, or between about 18 to 25residues in length.
 6. A composition comprising the siRNA molecule ofclaim 1 and a pharmaceutically acceptable carrier.
 7. A method ofactivating target-specific RNA interference (RNAi) in a cell or organismcomprising introducing into said cell or organism the siRNA of claim 1,said siRNA being introduced in an amount sufficient for degradation oftarget mRNA to occur, thereby activating target-specific RNAi in thecell.
 8. A cell or organism obtained by the method of claim
 7. 9. A kitcomprising reagents for activating target-specific RNA interference(RNAi) in a cell or organism, said kit comprising: (a) the siRNAmolecule of claim 1; and (b) instructions for use.
 10. The siRNA ofclaim 5, wherein the 2′-fluoro modified ribonucleotide comprises a2′-fluoro uridine or a 2′-fluoro cytidine.
 11. The siRNA of claim 3,wherein the 2′-deoxy ribonucleotide comprises at least one 2′-deoxyadenosine, at least one 2′-deoxy guanosine, or a mixture thereof. 12.The siRNA of claim 3, wherein the backbone-modified nucleotide comprisesa phosphorothioate.